Adenoviral vectors encoding hepatitis b viral antigens fused to herpes virus glycoprotein d and methods of using the same

ABSTRACT

Provided herein are non-naturally occurring variants of the hepatitis B virus (HBV) Core protein, the HBV polymerase N-terminal domain, and the HBV polymerase C-terminal domain, as well as immunogenic fragments thereof. Fusion proteins comprising the HBV variants fused to a herpes simplex virus (HSV) glycoprotein (gD) sequence, as well as methods of using the fusion proteins, are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/459,313, filed Aug. 27, 2021, which is a continuation ofInternational Application No. PCT/US2021/012630, filed on Jan. 8, 2021,which claims priority to U.S. Provisional Application No. 62/958,809,filed Jan. 9, 2020, U.S. Provisional Application No. 62/958,827, filedJan. 9, 2020, U.S. Provisional Application No. 62/967,242, filed Jan.29, 2020, U.S. Provisional Application No. 62/967,104, filed Jan. 29,2020, U.S. Provisional Application No. 63/064,506, filed Aug. 12, 2020,U.S. Provisional Application No. 63/064,571, filed Aug. 12, 2020, U.S.Provisional Application No. 63/112,202, filed Nov. 11, 2020, and U.S.Provisional Application No. 63/112,219, filed Nov. 11, 2020, thedisclosure of each of which is hereby incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on Jan. 7, 2021, isnamed 111876_000038_SL.txt and is 148 KB in size.

FIELD OF THE INVENTION

Disclosed herein are non-naturally occurring variants of the hepatitis Bvirus (HBV) Core protein, the HBV polymerase N-terminal domain, and theHBV polymerase C-terminal domain, as well as immunogenic fragmentsthereof and fusion proteins comprising the same.

BACKGROUND OF THE INVENTION

The World Health Organization estimates that, in 2015, 257 millionpeople were living with chronic hepatitis B infection (defined ashepatitis B surface antigen positive) and that hepatitis B resulted inan estimated 887,000 deaths, mostly from cirrhosis and hepatocellularcarcinoma (i.e., primary liver cancer). Assuming that women ofreproductive age constitute 25.3% of the world's population (UnitedNations data), adults chronically infected may include 65 million womenof childbearing age who can potentially transmit HBV to their babies(WHO Global Hepatitis Report 2017. Available at:apps_who_int/iris/bitstream/handle/10665/255016/9789241565455-eng.pdf;jsessionid=D78616700ED7322D4109CA4541FB94EA?sequence=1).The overall incidence rate in 2016 was 1.0 case per 100,000 population(Centers for Disease Control and Prevention. Viral HepatitisSurveillance—United States, 2017. Atlanta: US Department of Health andHuman Services, Centers for Disease Control and Prevention; 2019.Available at:www_cdc_gov/hepatitis/statistics/2017surveillance/index.htm.). In 2017alone, a total of 3,407 cases of acute hepatitis B were reported to theCenters for Disease Control and Prevention (CDC).

Despite the availability of a prophylactic HBV vaccine, the burden ofchronic HBV infection continues to be a significant unmet worldwidemedical problem, due to suboptimal treatment options and sustained ratesof new infections in most parts of the developing world.

SUMMARY OF THE INVENTION

Provided herein is a hepatitis B virus (HBV) Core protein comprising theamino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof.

Also provided is an HBV polymerase N-terminal domain comprising theamino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof.

An HBV polymerase C-terminal domain comprising the amino acid sequenceof SEQ ID NO: 10 or an immunogenic fragment thereof is also disclosed.

Fusion proteins comprising: an N-terminal herpes simplex virus (HSV)glycoprotein (gD) sequence or a variant thereof, the disclosed HBV Coreprotein, HBV polymerase N-terminal domain, HBV polymerase C-terminaldomain, or immunogenic fragments thereof, and a C-terminal HSV gDsequence or a variant thereof are also provided.

Also provided herein are fusion proteins comprising: an N-terminalherpes simplex virus (HSV) glycoprotein (gD) sequence or a variantthereof, combinations of the disclosed HBV Core protein, HBV polymeraseN-terminal domain, HBV polymerase C-terminal domain, and/or immunogenicfragments thereof, and a C-terminal HSV gD sequence or a variantthereof.

Nucleic acid molecules encoding the disclosed proteins or fusionproteins, vectors comprising the nucleic acid molecules, and vaccinescomprising the disclosed vectors are disclosed herein.

Also provided herein are methods of inducing an immune response to HBVin a subject, the method comprising providing to the subject aneffective amount of any of the disclosed fusion proteins, nucleic acidmolecules, vectors, or vaccines to thereby induce an immune response toHBV.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is furtherunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the disclosed proteins, vaccines, and methods,there are shown in the drawings exemplary embodiments of the proteins,vaccines, and methods; however, the proteins, vaccines, and methods arenot limited to the specific embodiments disclosed. In the drawings:

FIG. 1 illustrates the frequency of epitope-optimized Core amino acids.Amino acid residues are indicated on the X-axis; percent sequencesimilarity across all genomes analyzed is indicated on the Y-axis.

FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F illustratevaccine insert-specific T cell frequencies in C57Bl/6 mice followingintramuscular (i.m.) injection with the indicated doses of:replication-defective adenovirus vector of chimpanzee serotype 6 (AdC6)containing the epitope-optimized Core sequence genetically fused into gD(SEQ ID NO: 15) (AdC6-gDCore) (FIG. 2A and FIG. 2D); AdC6 containing theepitope-optimized polymerase C-terminal domain sequence geneticallyfused into gD (SEQ ID NO: 19) (AdC6-gDPolC) (FIG. 2B and FIG. 2E); andAdC6 containing the epitope-optimized polymerase N-terminal domainsequence genetically fused into gD (SEQ ID NO: 17) (AdC6-gDPolN) (FIG.2C and FIG. 2F). Mice were bled 14 days after the injection and T cellfrequencies to the various HBV inserts were analyzed by intracellularcytokine staining (ICS) for interferon (IFN)-γ upon stimulation of cellswith overlapping peptides representing the HBV sequences. Control cellswere cultured without peptides. Graphs show results for individual micewith medians indicated by the lines. FIG. 2A-2C show insert-specificCD8+ T cell frequency; FIG. 2D-2F show insert-specific CD4+ T cellfrequency.

FIG. 3A, FIG. 3B, and FIG. 3C illustrate T cell frequencies in differentmouse strains (A: C57Bl/6 mice; B: BALB/c mice; C: HLA-A2 transgenic(tg) mice) to pools of peptides representing the indicated HBV sequence.Results were obtained with splenocytes harvested 4 weeks afterimmunization and tested by ICS for IFN-γ. Peptides were arranged inmatrices so that recognition of 2 pools identified one peptide. Thegraphs show responses to the different pools; responses to a poolcontaining all peptides are shown to the right. Background frequenciesobtained without the peptides were subtracted. Pools that were deemed toelicit a response and peptides identified in response to different poolsare listed at the bottom of each figure. CD8+ T cell and CD4+ T cellresponses are shown for BALB/c mice; CD8+ T cell responses are shown forHLA-A2 tg mice, which carry a human MHC class I molecule but mouse MHCclass II molecules. T cells were gated on activated CD44+ cells. Eachconsecutively numbered “peptide” consists of 15 amino acids beginning onthe 1^(st), 6^(th), 11^(th), etc. amino acid of the Core, PolN, or PolCsequence. Thus, for example, peptide 1 of Core corresponds to aminoacids 1-15 of SEQ ID NO: 6 (i.e. the epitope-optimized Core amino acidsequence), peptide 2 of Core corresponds to amino acids 6-20 of SEQ IDNO: 6, peptide 3 of Core corresponds to amino acids 11-25 of SEQ ID NO:6, etc. Similarly, peptide 1 of PolN corresponds to amino acids 1-15 ofSEQ ID NO: 8 (ie. the epitope-optimized PolN amino acid sequence),peptide 2 of PolN corresponds to amino acids 6-20 of SEQ ID NO: 8,peptide 3 of PolN corresponds to amino acids 11-25 of SEQ ID NO: 8, etc.Likewise, peptide 1 of PolC corresponds to amino acids 1-15 of SEQ IDNO: 10 (i.e. the epitope-optimized PolC amino acid sequence), peptide 2of PolC corresponds to amino acids 6-20 of SEQ ID NO: 10, peptide 3 ofPolC corresponds to amino acids 11-25 of SEQ ID NO: 10, etc.

FIG. 4A, FIG. 4B and FIG. 4C show the IFN-γ response upon boosting withAdC6-gDCore (A), AdC6-gDPolC (B), and AdC6-gDPolN (C) in C57Bl/6 miceimmunized with various doses of the indicated vectors. The left graphsshow responses tested from blood 2 weeks after priming with AdC6 vector.Mice were boosted 8 weeks later with the same doses of AdC7 vectorsexpressing the same inserts. The right graphs show responses at 2 weeksafter the boost in blood.

FIG. 5A, FIG. 5B, and FIG. 5C illustrate T cell frequencies in differentmouse strains (A: C57Bl/6 mice; B: BALB/c mice; C: HLA-A2 tg mice) topools of peptides representing the indicated HBV sequence. Mice wereprimed with AdC6 vectors expressing either of the 3 inserts (i.e., Core,PolC, or PolN) and were boosted 8 weeks later with AdC7 vectorsexpressing the same inserts. Results were obtained with splenocytesharvested 4 weeks after the immunization and tested by ICS for IFN-γ.Peptides were arranged in matrices so that recognition of 2 poolsidentified one peptide. The graphs show responses to the differentpools; responses to a pool containing all peptides are shown to theright. Background frequencies obtained without the peptides weresubtracted. Pools that were deemed to elicit a response and peptidesidentified in response to different pools are listed at the bottom ofeach figure. CD8+ T cell and CD4+ T cell responses are shown for BALB/cmice; CD8+ T cell responses are shown for HLA-A2 tg mice which carry ahuman MHC class I molecule but mouse MHC class II molecules. T cellswere gated on activated CD44+ cells. Each consecutively numbered“peptide” consists of 15 amino acids beginning on the 1^(st), 6^(th),11^(th), etc. amino acid of the Core, PolN, or PolC sequence. Thus, forexample, peptide 1 of Core corresponds to amino acids 1-15 of SEQ ID NO:6 (i.e. the epitope-optimized Core amino acid sequence), peptide 2 ofCore corresponds to amino acids 6-20 of SEQ ID NO: 6, peptide 3 of Corecorresponds to amino acids 11-25 of SEQ ID NO: 6, etc. Similarly,peptide 1 of PolN corresponds to amino acids 1-15 of SEQ ID NO: 8 (i.e.the epitope-optimized PolN amino acid sequence), peptide 2 of PolNcorresponds to amino acids 6-20 of SEQ ID NO: 8, peptide 3 of PolNcorresponds to amino acids 11-25 of SEQ ID NO: 8, etc. Likewise, peptide1 of PolC corresponds to amino acids 1-15 of SEQ ID NO: 10 (i.e. theepitope-optimized PolC amino acid sequence), peptide 2 of PolCcorresponds to amino acids 6-20 of SEQ ID NO: 10, peptide 3 of PolCcorresponds to amino acids 11-25 of SEQ ID NO: 10, etc.

FIG. 6 illustrates the effect of vaccination on HBV genome copy numbersin serum upon AAV-1.3HBV challenge. A group of 3 mice were challengedwith 1×10¹⁰, 1×10¹¹ or 1.5×10¹¹ virus genomes (vg) of anadeno-associated virus 8 (AAV8)-1.3HBV vector and 8 weeks later werevaccinated with AdC6-gDPolN. Viral titers were tested 8 weeks aftervaccination and compared to pre-vaccination titers. Viral changes frombaseline for each treatment group are shown.

FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, and FIG. 7E illustrate exemplary HBVepitope shifting experiments. FIG. 7A—mice were immunized with theAdC6-gDPolN vaccine. Four weeks later splenocytes were tested byintracellular cytokine staining for IFN-γ responses to peptide poolsrepresenting the PolN sequence. T cells after stimulation were stainedfor T cell markers. FIG. 7B—results obtained with the same assay usingsplenocytes from mice that were challenged with 1×10¹⁰ vg ofAAV8-1.3-HBV. Mice were vaccinated 4 weeks later and T cell responseswere tested from spleens 10 weeks later. FIG. 7C—results obtained withthe same assay using splenocytes from mice that had been challenged with1.5×10¹¹ vg of AAV8-1.3-HBV. Mice were vaccinated 4 weeks later and Tcell responses were tested from spleen 10 weeks later. FIGS. 7A, 7B, and7C show the frequencies of IFN-γ producing CD44+CD8+ T cells over allCD44+CD8+ T cells. Background responses obtained by splenocytesincubated without peptide pools were subtracted. FIG. 7D—peptide pools.FIG. 7E—individual peptide sequences. FIG. 7E discloses SEQ ID NOs:55-68 and 189-233, respectively, in order of appearance.

FIG. 8A, FIG. 8B, and FIG. 8C show data from the same experimentdescribed above in FIG. 7. Based on the responses to the peptide pools,it was determined which individual peptides (both pools and peptidesshown in FIG. 7) were positive. The graphs show responses to all of thepeptides. Each peptide was present in two pools and therefore two valuesfor frequencies were obtained for each peptide; only the lower datapoints are shown in this figure.

FIG. 9A, FIG. 9B, and FIG. 9C illustrate the results from exemplaryimmunogenicity experiments performed on C57Bl/6 mice (n=5 per group)injected with various doses of exemplary AdC6-gDCore, AdC6-gDPolN, orAdC6-gDPolC vectors and boosted with AdC7 vectors containing the sameinsert (i.e. AdC7-gDCore, AdC7-gDPolN, or AdC7-gDPolC vectors) twomonths after the first injection. FIG. 9A illustrates antigenimmunogenicity, FIG. 9B illustrates the duration of response, and FIG.9C illustrates the prime-boost response.

FIG. 10 illustrates the CD8+ T cell peptide recognition of PolN epitopesin BALB/c, C57Bl/6, and HLA-A2 transgenic mice after vaccination with aprime of AdC6-gDPolN and a boost of AdC7-gDPolN. CD8+ T cell peptiderecognition was calculated as the fraction of positive peptidesrecognized two weeks after either the prime or the boost by the totalnumber of overlapping 8 peptides from PolN (59 peptides total).

FIG. 11A and FIG. 11B illustrate vaccine-induced HBV-specific CD8+ Tcell response in the liver of C57Bl/6 mice injected with the indicatedvectors. *p-value between 0.01-0.05; ***p-value between 0.0001-0.001;via 1-way ANOVA.

FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E, and FIG. 12Fillustrate hematoxylin & eosin staining of liver samples from C57Bl/6mice injected with the indicated vectors. 20× magnification. Arrowsindicate areas of lymphocytic infiltrates.

FIG. 13A and FIG. 13B illustrate vaccine-induced markers of CD8+ T cellactivation/exhaustion in the liver of C57Bl/6 mice injected with theindicated vectors. **p-value between 0.001-0.01; ***p-value between0.0001-0.001; via 1-way ANOVA.

FIG. 14A and FIG. 14B illustrate HBV viral dynamics in C57Bl/6 miceinjected with an exemplary AdC6-gDPolN vector. The median HBV DNA VL/mlat week 4-7.3 log₁₀ cps/mL are provided. n=7; one mouse excluded formissing data.

FIG. 15A and FIG. 15B illustrate the impact of AAV-induced HBV on CD8+ Tcell responses in C57Bl/6 mice first injected with 10¹⁰ or 10¹¹ vg ofAAV-1.3HBV and then four weeks later boosted with 10¹⁰ vp of anexemplary AdC6-gDPolN vector. In FIG. 15B, each slice represents anindividual epitope with size showing the proportion of the total; onlyresponses >0.1% were included. Pullouts represent epitopes onlyrecognized in AAV8-1.3HBV infected mice.

FIG. 16 illustrates the frequencies of IFN-γ-producing CD8+ T cells forindividual C57Bl/6 mice that were injected i.v. with the 10¹⁰ vg of theAAV8-1.3HBV vectors, vaccinated 4 weeks later with 5×10⁹ vp of theAdC6-gDPolN vector, and boosted 2 months later with the same dose of theAdC7-gDPolN vaccine. Control mice only received the vaccine. Naïve miceserved as additional controls.

FIG. 17A and FIG. 17B illustrate: A) % of CD8⁺ T cells within thelymphatic infiltrates of livers of individual mice; and B) thefrequencies of PolN-tetramer⁺CD8⁺ T cells within the same infiltrates.C57Bl/6 mice were injected i.v. with the 10¹⁰ or 10¹¹ vg of theAAV8-1.3HBV vector, were vaccinated 4 weeks later with 5×10⁹ vp of theAdC6-gDPolN vector, and were boosted 2 months later with the same doseof the AdC7-gDPolN vaccine. Control mice only received the vaccine.Naïve mice served as additional controls.

FIG. 18A, FIG. 18B, FIG. 18C, FIG. 18D, FIG. 18E, and FIG. 18Fillustrate the phenotypes of the infiltrating tetramer⁺CD8⁺ T cells incomparison to naïve (i.e., tetramer⁻ CD44⁻ CD8⁺) T cells analyzed withthe mean fluorescent intensity (MFI) of the indicated markers. Lineswith stars above indicate significant differences by multiple t-test.(*) p≤0.05-0.01, (**) p≤0.01-0.001, (***) p≤0.001-0.0001, (****)p≤0.0001.

FIG. 19A, FIG. 19B, FIG. 19C, FIG. 19D, FIG. 19E, and FIG. 19Fillustrate the percentage of Tet⁺ or naïve CD8⁺ T cells positive for theindicated markers. Lines with stars above indicate significantdifferences by multiple t-test. (*) p≤0.05-0.01, (**) p≤0.01-0.001,(***) p≤0.001-0.0001, (****) p≤0.0001.

FIG. 20A, FIG. 20B, FIG. 20C, FIG. 20D, FIG. 20E, and FIG. 20Fillustrate the CD8⁺ T cell response to individual peptides spanning thePolN sequence. Total pool—response to mixtures of all PolN peptides;Naïve—response of naïve mice to mixtures of all PolN peptides. FIG. 20Aand FIG. 20D show CD8⁺ T cell responses of mice that received just theAdC6-gDPolN vaccine. FIG. 20B and FIG. 20E show CD8⁺ T cell responses ofmice that were injected with 10¹⁰ vg of AAV8-1.3HBV 4 weeks prior tovaccination with AdC6-gDPolN. FIG. 20C and FIG. 20F show CD8⁺ T cellresponses of mice that were injected with 10¹¹ vg of AAV8-1.3HBV 4 weeksprior to vaccination with AdC6-gDPolN. FIGS. 20A, 20B and 20C can beused to calculate the breadth of the immune response by individualepitopes using the peptide pools shown in FIG. 7D and the individualpeptide sequences recognized using FIG. 7E.

FIG. 21A and FIG. 21B illustrate the PolN-specific CD8+ T cells in thespleen or liver of mice. FIG. 21A left panel shows CD8⁺ T cell responsesin spleen of AAV8-1.3HBV injected mice that did or did not receive theAdC6-gDPolN vaccine at 5×10¹⁰ vp subsequently. FIG. 21A middle panelshows CD8⁺ T cell frequencies in livers of mice that were treated withdifferent doses of AAV8-1.3HBV and then received vaccines in a primeboost regimen. FIG. 21A right panel shows the levels of Tox-1 expressionin PolN-specific CD8⁺ T cells or naïve CD8⁺ T cells from the sameexperiment. FIG. 21B illustrates % IFN-γ⁺CD8⁺ T cells.

FIG. 22A and FIG. 22B illustrate A) CD8+ T cell frequencies in the bloodof mice injected with the indicated AdC6 vectors; and B) frequencies oftetramer+CD8+ T cells.

FIG. 23 illustrates CD8+ T cell frequencies in the blood of miceinjected with the indicated AdC7 vectors.

FIG. 24A, FIG. 24B, FIG. 24C, FIG. 24D, FIG. 24E, and FIG. 24Fillustrate CD8⁺ (FIG. 24A-FIG. 24C) and CD4+(FIG. 24D-FIG. 24F) T cellfrequencies to the gDHBV2 and gDHBV3 inserts in blood of mice injectedwith the indicated AdC7 vectors (“after prime”) and then boosted withthe corresponding AdC6 vectors (“after boost”). Graphs show frequenciesof T cells producing IFN-γ, frequencies of T cells producing TNF-α, andthe sum of frequencies of T cells producing either cytokine.

FIG. 25A and FIG. 25B illustrate the HBV DNA viral titer in C57Bl/6 micethat were challenged with 1×10⁹ vg of AAV8-1.3HBV and were vaccinated 4weeks later with 1×10¹⁰ vp of AdC6-gDPolN (“gDPolN”), AdC6-gDHBV2(“gDHBV2”), AdC6-gDHBV3 (“gDHBV3”), or AdC6-HBV2 without gD (“HBV2”);AAV-infected, non-vaccinated animals (“naïve”), and non-AAV-infected,non-vaccinated animals (data not shown) served as controls. FIG. 25Aillustrates the viral titer for each group at weeks 4 and 8 after AAVchallenge; FIG. 25B illustrates the results of the individual mice atweeks 4 and 8 after AAV challenge.

FIG. 26A, FIG. 26B, FIG. 26C, and FIG. 26D illustrate the percent ofparental IFN-γ and/or TNF-α producing CD8⁺ T cells (FIG. 26A), CD44+CD8+T cells (FIG. 26B), CD4+ T cells (FIG. 26C) or CD44+CD4+ T cells (FIG.26D) two and eight weeks after prime and two and four weeks after theboost (as the mean) using the indicated construct.

FIG. 27A, FIG. 27B, and FIG. 27C illustrate CD8⁺ T cells at multipletime points: four weeks after prime (FIG. 27A); two weeks after theboost (FIG. 27B); and four weeks after the boost (FIG. 27C) with theindicated constructs (PolN=gDPolN; HBV2=gDHBV2; HBV3=gDHBV3). The graphshows the overall frequencies of CD8⁺ T cells producing IFN-γ⁺ asassessed by ICS.

FIG. 28A, FIG. 28B, and FIG. 28C illustrate cytokine-producing CD4+ Tcells at multiple time points: four weeks after prime (FIG. 28A); twoweeks after the boost (FIG. 28B); and four weeks after the boost (FIG.28C) with the indicated constructs (PolN=gDPolN; HBV2=gDHBV2;HBV3=gDHBV3) as assessed by ICS. The dashed line indicates the cut-offfor positive responses, based on the results from the naïve mice.

FIG. 29A and FIG. 29B illustrate the results of tetramer staining gatedon either CD8+ T cells (FIG. 29A) or CD44+CD8+ T cells (FIG. 29B) atfour weeks after the prime with the indicated construct (PolN=gDPolN;HBV2=gDHBV2).

FIG. 30A, FIG. 30B, FIG. 30C, FIG. 30D, FIG. 30E, and FIG. 30Fillustrates the phenotypes of the tetramer+CD8+ T cells shown as themean fluorescent intensity of a dye linked to the indicated antibody:FIG. 30A—anti-PD1 antibody conjugated to BV605; FIG. 30B—anti-LAG3antibody conjugated to BV650; FIG. 30C—anti-TIM3 antibody conjugated toPe-Cy7-A; FIG. 30D—anti-CTLA4 antibody conjugated to PE-A; FIG.30E—anti-EOMES antibody conjugated to AF488; and FIG. 30F—anti-T-betantibody conjugated to BV786.

FIG. 31 illustrates the CD8⁺ T cell responses after a prime vaccinationof 5×10¹⁰ vp AdC7-gDHBV2 followed two months later by vaccination with5×10¹⁰ vp AdC6-gDHBV2. Numbers on the X axis correspond to the SEQ ID NOas provided herein.

FIG. 32 illustrates the CD8+ T cell responses after a prime vaccinationwith 5×10⁹ vp AdC7-gDHBV2 followed two months later by vaccination with5×10⁹ vp AdC6-gDHBV2. Numbers on the X axis correspond to the SEQ ID NOas provided herein.

FIG. 33 shows the immunogenicity after a prime vaccination with 5×10¹⁰vp AdC7-gDHBV3 followed two months later by vaccination with 5×10¹⁰ vpAdC6-gDHBV3. Numbers on the X axis correspond to the SEQ ID NO asprovided herein.

FIG. 34 illustrates the immunogenicity of the AdC6-gDHBV2 andAdC7-gDHBV2 vaccines corresponding to the SEQ ID NO (X axis) as providedherein. Core, PolC, and PolN regions in both HBV2 constructs wereimmunogenic.

FIG. 35 illustrates the immunogenicity of the AdC6-gDHBV3 andAdC7-gDHBV3 vaccines corresponding to the SEQ ID NO (X axis) as providedherein. Core, PolC, and PolN regions in both HBV3 constructs wereimmunogenic.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosed proteins, vaccines, and methods may be understood morereadily by reference to the following detailed description taken inconnection with the accompanying figures, which form a part of thisdisclosure. It is to be understood that the disclosed proteins,vaccines, and methods are not limited to the specific proteins,vaccines, and methods described and/or shown herein, and that theterminology used herein is for the purpose of describing particularembodiments by way of example only and is not intended to be limiting ofthe claimed proteins, vaccines, and methods.

Unless specifically stated otherwise, any description as to a possiblemechanism or mode of action or reason for improvement is meant to beillustrative only, and the disclosed proteins, vaccines, and methods arenot to be constrained by the correctness or incorrectness of any suchsuggested mechanism or mode of action or reason for improvement.

Throughout this text, the descriptions refer to proteins and methods ofusing said proteins. Where the disclosure describes or claims a featureor embodiment associated with a proteins, such a feature or embodimentis equally applicable to the methods of using said proteins. Likewise,where the disclosure describes or claims a feature or embodimentassociated with a method of using the proteins, such a feature orembodiment is equally applicable to the proteins.

Where a range of numerical values is recited or established herein, therange includes the endpoints thereof and all the individual integers andfractions within the range, and also includes each of the narrowerranges therein formed by all the various possible combinations of thoseendpoints and internal integers and fractions to form subgroups of thelarger group of values within the stated range to the same extent as ifeach of those narrower ranges was explicitly recited. Where a range ofnumerical values is stated herein as being greater than a stated value,the range is nevertheless finite and is bounded on its upper end by avalue that is operable within the context of the invention as describedherein. Where a range of numerical values is stated herein as being lessthan a stated value, the range is nevertheless bounded on its lower endby a non-zero value. It is not intended that the scope of the inventionbe limited to the specific values recited when defining a range. Allranges are inclusive and combinable.

When values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms anotherembodiment. Reference to a particular numerical value includes at leastthat particular value, unless the context clearly dictates otherwise.

It is to be appreciated that certain features of the disclosed proteins,vaccines, and methods which are, for clarity, described herein in thecontext of separate embodiments, may also be provided in combination ina single embodiment. Conversely, various features of the disclosedproteins, vaccines, and methods that are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany subcombination.

As used herein, the singular forms “a,” “an,” and “the” include theplural.

Various terms relating to aspects of the description are used throughoutthe specification and claims. Such terms are to be given their ordinarymeaning in the art unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinitions provided herein.

As used herein, “immunogenic fragment thereof” refers to a portion ofthe disclosed HBV Core (Core), HBV polymerase N-terminal domain (PolN),or HBV polymerase C-terminal domain (PolC) that can produce an immuneresponse in a subject.

As used herein, “providing to the subject” and similar terms indicate aprocedure by which the fusion proteins, nucleic acid molecules, vectors,or vaccines are delivered to a subject such that target cells, tissues,or segments of the body of the subject are contacted with the fusionproteins, nucleic acid molecules, vectors, or vaccines. “Providing tothe subject” includes parenteral and non-parenteral routes ofadministration.

The term “biosimilar” (of an approved reference product/biological drug,i.e., reference listed drug) refers to a biological product that ishighly similar to the reference product notwithstanding minordifferences in clinically inactive components with no clinicallymeaningful differences between the biosimilar and the reference productin terms of safety, purity and potency, based upon data derived from (a)analytical studies that demonstrate that the biological product ishighly similar to the reference product notwithstanding minordifferences in clinically inactive components; (b) animal studies(including the assessment of toxicity); and/or (c) a clinical study orstudies (including the assessment of immunogenicity and pharmacokineticsor pharmacodynamics) that are sufficient to demonstrate safety, purity,and potency in one or more appropriate conditions of use for which thereference product is licensed and intended to be used and for whichlicensure is sought for the biosimilar. The biosimilar may be aninterchangeable product that may be substituted for the referenceproduct at the pharmacy without the intervention of the prescribinghealthcare professional. To meet the additional standard of“interchangeability,” the biosimilar is to be expected to produce thesame clinical result as the reference product in any given patient and,if the biosimilar is administered more than once to an individual, therisk in terms of safety or diminished efficacy of alternating orswitching between the use of the biosimilar and the reference product isnot greater than the risk of using the reference product without suchalternation or switch. The biosimilar utilizes the same mechanisms ofaction for the proposed conditions of use to the extent the mechanismsare known for the reference product. The condition or conditions of useprescribed, recommended, or suggested in the labeling proposed for thebiosimilar have been previously approved for the reference product. Theroute of administration, the dosage form, and/or the strength of thebiosimilar are the same as those of the reference product and thebiosimilar is manufactured, processed, packed or held in a facility thatmeets standards designed to assure that the biosimilar continues to besafe, pure and potent. The biosimilar may include minor modifications inthe amino acid sequence when compared to the reference product, such asN- or C-terminal truncations that are not expected to change thebiosimilar performance. Biosimilars of the disclosed proteins and fusionproteins are included within the scope of this disclosure.

The term “subject” as used herein is intended to mean any animal, inparticular, mammals. Although induction of an immune response in mice isexemplified herein, any type of mammal can be treated using thedisclosed methods. Thus, the methods are applicable to human andnonhuman animals, although preferably used with mice and humans, andmost preferably with humans.

The term “comprising” is intended to include examples encompassed by theterms “consisting essentially of” and “consisting of”; similarly, theterm “consisting essentially of” is intended to include examplesencompassed by the term “consisting of.”

The following abbreviations are used herein: hepatitis B virus (HBV);adenovirus (Ad); herpes simplex virus (HSV); glycoprotein (gD); andvirus genomes (vg).

Provided herein is a non-naturally occurring variant of the hepatitis Bvirus (HBV) Core protein. The disclosed HBV Core protein can comprisethe amino acid sequence of SEQ ID NO: 6 or an immunogenic fragmentthereof. Exemplary immunogenic fragments of SEQ ID NO: 6 include SEQ IDNOs: 20-54 provided in Table 3, below. In some embodiments, theimmunogenic fragment of the HBV Core protein comprises the amino acidsequence of SEQ ID NO: 180. In some embodiments, the immunogenicfragment of the HBV Core protein comprises the amino acid sequence ofSEQ ID NO: 183.

Nucleic acid molecules encoding the HBV Core protein or an immunogenicfragment thereof are also provided. The nucleic acid molecule can encodethe HBV Core protein comprising the amino acid sequence of SEQ ID NO: 6.In some embodiments, the nucleic acid molecule comprises the nucleotidesequence of SEQ ID NO: 7. The nucleic acid molecules can encode the Corefragments provided in Table 3. In some embodiments, the nucleic acidmolecule encodes the amino acid sequence of SEQ ID NO: 180. In someembodiments, the nucleic acid molecule encodes the amino acid sequenceof SEQ ID NO: 183.

Vectors comprising the nucleic acid molecules encoding the HBV Coreprotein or an immunogenic fragment thereof are also provided. Suitablevectors include viral vectors, such as lentiviral vectors, retroviralvectors, adenoviral vectors, adeno-associated viral vectors, alphavirusreplicons, herpes virus vectors, pox virus vectors, and rhabdovirusvectors. In some embodiments, the viral vector is an adenoviral vector.The adenoviral vector can be a chimpanzee-derived adenoviral vector. Insome aspects, the vector is an AdC68 vector as described in Farina S F,Gao G P, Xiang Z Q, Rux J J, Burnett R M, Alvira M R, Marsh J, Ertl H C,Wilson J M. “Replication-defective vector based on a chimpanzeeadenovirus.” J Virol. 2001 December; 75(23):11603-13. In some aspects,the vector is an AdC7 vector as described in Reyes-Sandoval A,Fitzgerald J C, Grant R, Roy S, Xiang Z Q, Li Y, Gao G P, Wilson J M,Ertl H C. “Human immunodeficiency virus type 1-specific immune responsesin primates upon sequential immunization with adenoviral vaccinecarriers of human and simian serotypes” J Virol. 2004 July;78(14):7392-9. In some aspects, the vector is an AdC6 vector asdescribed in Pinto A R, Fitzgerald J C, Giles-Davis W, Gao G P, Wilson JM, Ertl H C. “Induction of CD8+ T cells to an HIV-1 antigen through aprime boost regimen with heterologous El-deleted adenoviral vaccinecarriers” J Immunol. 2003 Dec. 15; 171(12):6774-9.

In some embodiments, the vector comprises the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 7. In some embodiments,the vector is an AdC6 vector comprising the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 7. In some embodiments,the vector is an AdC7 vector comprising the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 7.

In some embodiments, the vector comprises the nucleic acid molecule thatencodes the amino acid sequence of SEQ ID NO: 180. In some aspects, thevector is an AdC6 vector. In some aspects, the vector is an AdC7 vector.In some embodiments, the vector comprises the nucleic acid molecule thatencodes the amino acid sequence of SEQ ID NO: 183. In some aspects, thevector is an AdC6 vector. In some aspects, the vector is an AdC7 vector.

Vaccines comprising the vectors comprising the nucleic acid moleculesencoding the HBV Core protein or an immunogenic fragment thereof arealso disclosed. In some embodiments, the vaccine comprises a vectorcomprising the nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO: 7. In some embodiments, the vaccine comprises an AdC6vector comprising the nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO: 7. In some embodiments, the vaccine comprises anAdC7 vector comprising the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 7. In some embodiments, the vaccinecomprises an AdC6 vector comprising the nucleic acid molecule thatencodes the amino acid sequence of SEQ ID NO: 180. In some embodiments,the vaccine comprises an AdC7 vector comprising the nucleic acidmolecule that encodes the amino acid sequence of SEQ ID NO: 180. In someembodiments, the vaccine comprises an AdC6 vector that comprises thenucleic acid molecule that encodes the amino acid sequence of SEQ ID NO:183. In some embodiments, the vaccine comprises an AdC7 vector thatcomprises the nucleic acid molecule that encodes the amino acid sequenceof SEQ ID NO: 183.

The vaccine can further comprise a pharmaceutically acceptable carrieror pharmaceutical acceptable excipient. As used herein,“pharmaceutically acceptable carrier” or “pharmaceutical acceptableexcipient” includes any material which, when combined with the disclosedfusion proteins, nucleic acids, or vectors, allows the fusion proteins,nucleic acids, or vectors to retain biological activity and isnon-reactive with the subject's immune system. Examples include, but arenot limited to, any of the standard pharmaceutical carriers such as aphosphate buffered saline solution, water, emulsions such as oil/wateremulsion, and various types of wetting agents. Preferred diluents foraerosol or parenteral administration are phosphate buffered saline ornormal (0.9%) saline. Compositions comprising such carriers areformulated by well-known conventional methods (see, for example,Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., MackPublishing Co., Easton, Pa., 1990; and Remington, The Science andPractice of Pharmacy 20th Ed. Mack Publishing, 2000).

Also disclosed herein are non-naturally occurring variants of the HBVpolymerase N-terminal domain (PolN) and the HBV polymerase C-terminaldomain (PolC). The disclosed HBV polymerase N-terminal domain cancomprise the amino acid sequence of SEQ ID NO: 8 or an immunogenicfragment thereof. Exemplary immunogenic fragments of SEQ ID NO: 8include SEQ ID NOs: 55-113 provided in Table 4, below. In someembodiments, the immunogenic fragment of the HBV PolN comprises theamino acid sequence of SEQ ID NO: 178. In some embodiments, theimmunogenic fragment of the HBV PolN comprises the amino acid sequenceof SEQ ID NO: 181. The disclosed HBV polymerase C-terminal domain cancomprise the amino acid sequence of SEQ ID NO: 10 or an immunogenicfragment thereof. Exemplary immunogenic fragments of SEQ ID NO: 10include SEQ ID NOs: 114-172 provided in Table 5, below. In someembodiments, the immunogenic fragment of the HBV PolC comprises theamino acid sequence of SEQ ID NO: 179. In some embodiments, theimmunogenic fragment of the HBV PolC comprises the amino acid sequenceof SEQ ID NO: 182.

Nucleic acid molecules encoding the HBV polymerase N-terminal domain oran immunogenic fragment thereof, or the HBV polymerase C-terminal domainor an immunogenic fragment thereof, are also provided. The nucleic acidmolecule can encode the HBV polymerase N-terminal domain comprising theamino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleicacid molecule encoding the HBV polymerase N-terminal domain comprisesthe nucleotide sequence of SEQ ID NO: 9. The nucleic acid molecules canencode the HBV polymerase N-terminal domain fragments provided in Table4. The nucleic acid molecule can encode the HBV polymerase C-terminaldomain comprising the amino acid sequence of SEQ ID NO: 10. In someembodiments, the nucleic acid molecule encoding the HBV polymeraseC-terminal domain comprises the nucleotide sequence of SEQ ID NO: 11.The nucleic acid molecules can encode the HBV polymerase C-terminaldomain fragments provided in Table 5. In some embodiments, the nucleicacid molecule encodes the amino acid sequence of SEQ ID NO: 178. In someembodiments, the nucleic acid molecule encodes the amino acid sequenceof SEQ ID NO: 181. In some embodiments, the nucleic acid moleculeencodes the amino acid sequence of SEQ ID NO: 179. In some embodiments,the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:182.

Vectors comprising the nucleic acid molecules encoding the HBVpolymerase N-terminal domain or an immunogenic fragment thereof orC-terminal domain or an immunogenic fragment thereof are also provided.Suitable vectors include those described above. In some embodiments, thevector comprises the nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO: 9. In some embodiments, the vector comprises thenucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:11. In some aspects, the vector is an adenoviral vector. Suitableadenoviral vectors include, for example, an AdC6 vector or AdC7 vector.In some embodiments, the vector is an AdC6 vector comprising the nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO: 9. Insome embodiments, the vector is an AdC7 vector comprising the nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO: 9. Insome embodiments, the vector is an AdC6 vector comprising the nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO: 11. Insome embodiments, the vector is an AdC7 vector comprising the nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO: 11. Insome embodiments, the vector comprises the nucleic acid molecule thatencodes the amino acid sequence of SEQ ID NO: 178. In some aspects, thevector is an AdC6 vector. In some aspects, the vector is an AdC7 vector.In some embodiments, the vector comprises the nucleic acid molecule thatencodes the amino acid sequence of SEQ ID NO: 181. In some aspects, thevector is an AdC6 vector. In some aspects, the vector is an AdC7 vector.In some embodiments, the vector comprises the nucleic acid molecule thatencodes the amino acid sequence of SEQ ID NO: 179. In some aspects, thevector is an AdC6 vector. In some aspects, the vector is an AdC7 vector.In some embodiments, the vector comprises the nucleic acid molecule thatencodes the amino acid sequence of SEQ ID NO: 182. In some aspects, thevector is an AdC6 vector. In some aspects, the vector is an AdC7 vector.

Vaccines comprising the vectors comprising the nucleic acid moleculesencoding the HBV polymerase N-terminal domain or an immunogenic fragmentthereof or HBV polymerase C-terminal domain or an immunogenic fragmentthereof are also disclosed. In some embodiments, the vaccine comprises avector comprising the nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO: 9. The vaccine can comprise an AdC6 vectorcomprising the nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO: 9. The vaccine can comprise an AdC7 vector comprising thenucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:9. In some embodiments, the vaccine comprises a vector comprising thenucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:11. The vaccine can comprise an AdC6 vector comprising the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO: 11. Thevaccine can comprise an AdC7 vector comprising the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 11. The vaccine canfurther comprise a pharmaceutically acceptable carrier or pharmaceuticalacceptable excipient as disclosed above. In some embodiments, thevaccine comprises a vector comprising the nucleic acid molecule thatencodes the amino acid sequence of SEQ ID NO: 178. In some aspects, thevector is an AdC6 vector. In some aspects, the vector is an AdC7 vector.In some embodiments, the vaccine comprises a vector comprising thenucleic acid molecule that encodes the amino acid sequence of SEQ ID NO:181. In some aspects, the vector is an AdC6 vector. In some aspects, thevector is an AdC7 vector. In some embodiments, the vaccine comprises avector comprising the nucleic acid molecule that encodes the amino acidsequence of SEQ ID NO: 179. In some aspects, the vector is an AdC6vector. In some aspects, the vector is an AdC7 vector. In someembodiments, the vaccine comprises a vector comprising the nucleic acidmolecule that encodes the amino acid sequence of SEQ ID NO: 182. In someaspects, the vector is an AdC6 vector. In some aspects, the vector is anAdC7 vector.

Fusion proteins comprising combinations of the disclosed HBV Coreprotein or immunogenic fragments thereof, the HBV polymerase N-terminaldomain or immunogenic fragments thereof, and/or the HBV polymeraseC-terminal domain or immunogenic fragments thereof are also providedherein. For example, the fusion protein can comprise:

-   -   (1) an HBV Core protein comprising the amino acid sequence of        SEQ ID NO: 6 or an immunogenic fragment thereof and an HBV        polymerase N-terminal domain comprising the amino acid sequence        of SEQ ID NO: 8 or an immunogenic fragment thereof;    -   (2) one or more immunogenic fragments of the HBV Core protein        comprising the amino acid sequence of SEQ ID NO: 6 and one or        more immunogenic fragments of the HBV polymerase N-terminal        domain comprising the amino acid sequence of SEQ ID NO: 8. For        example, one or more of SEQ ID NOs: 20-54 provided in Table 3        (immunogenic fragments of SEQ ID NO: 6) and one or more of SEQ        ID NOs: 55-113 provided in Table 4 (immunogenic fragments of SEQ        ID NO: 8);    -   (3) an HBV Core protein comprising the amino acid sequence of        SEQ ID NO: 6 or an immunogenic fragment thereof and an HBV        polymerase C-terminal domain comprising the amino acid sequence        of SEQ ID NO: 10 or an immunogenic fragment thereof;    -   (4) one or more immunogenic fragments of the HBV Core protein        comprising the amino acid sequence of SEQ ID NO: 6 and one or        more immunogenic fragments of the HBV polymerase C-terminal        domain comprising the amino acid sequence of SEQ ID NO: 10. For        example, one or more of SEQ ID NOs: 20-54 provided in Table 3        (immunogenic fragments of SEQ ID NO: 6) and one or more of SEQ        ID NOs: 114-172 provided in Table 5 (immunogenic fragments of        SEQ ID NO: 10);    -   (5) an HBV polymerase N-terminal domain comprising the amino        acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof        and an HBV polymerase C-terminal domain comprising the amino        acid sequence of SEQ ID NO: 10 or an immunogenic fragment        thereof;    -   (6) one or more immunogenic fragments of the HBV polymerase        N-terminal domain comprising the amino acid sequence of SEQ ID        NO: 8 and one or more immunogenic fragments of the HBV        polymerase C-terminal domain comprising the amino acid sequence        of SEQ ID NO: 10. For example, one or more of SEQ ID NOs: 55-113        provided in Table 4 (immunogenic fragments of SEQ ID NO: 8) and        one or more of SEQ ID NOs: 114-172 provided in Table 5        (immunogenic fragments of SEQ ID NO: 10);    -   (7) an HBV Core protein comprising the amino acid sequence of        SEQ ID NO: 6 or an immunogenic fragment thereof, an HBV        polymerase N-terminal domain comprising the amino acid sequence        of SEQ ID NO: 8 or an immunogenic fragment thereof, and an HBV        polymerase C-terminal domain comprising the amino acid sequence        of SEQ ID NO: 10 or an immunogenic fragment thereof;    -   (8) one or more immunogenic fragments of the HBV Core protein        comprising the amino acid sequence of SEQ ID NO: 6, one or more        immunogenic fragments of the HBV polymerase N-terminal domain        comprising the amino acid sequence of SEQ ID NO: 8, and one or        more immunogenic fragments of the HBV polymerase C-terminal        domain comprising the amino acid sequence of SEQ ID NO: 10. For        example, one or more of SEQ ID NOs: 20-54 provided in Table 3        (immunogenic fragments of SEQ ID NO: 6), one or more of SEQ ID        NOs: 55-113 provided in Table 4 (immunogenic fragments of SEQ ID        NO: 8), and one or more of SEQ ID NOs: 114-172 provided in Table        5 (immunogenic fragments of SEQ ID NO: 10);    -   (9) An HBV polymerase N-terminal domain comprising the amino        acid sequence of SEQ ID NO: 178 or an immunogenic fragment        thereof, an HBV polymerase C-terminal domain comprising the        amino acid sequence of SEQ ID NO: 179 or an immunogenic fragment        thereof, and an HBV Core protein comprising the amino acid        sequence of SEQ ID NO: 180 or an immunogenic fragment thereof,        or    -   (10) An HBV polymerase N-terminal domain comprising the amino        acid sequence of SEQ ID NO: 181 or an immunogenic fragment        thereof, an HBV polymerase C-terminal domain comprising the        amino acid sequence of SEQ ID NO: 182 or an immunogenic fragment        thereof, and an HBV Core protein comprising the amino acid        sequence of SEQ ID NO: 183 or an immunogenic fragment thereof.

The fusion protein can comprise an HBV polymerase N-terminal domaincomprising the amino acid sequence of SEQ ID NO: 178 or an immunogenicfragment thereof, an HBV polymerase C-terminal domain comprising theamino acid sequence of SEQ ID NO: 179 or an immunogenic fragmentthereof, and an HBV Core protein comprising the amino acid sequence ofSEQ ID NO: 180 or an immunogenic fragment thereof. In some embodiments,the fusion protein comprises the amino acid sequence of SEQ ID NO: 174.

The fusion protein can comprise an HBV polymerase N-terminal domaincomprising the amino acid sequence of SEQ ID NO: 181 or an immunogenicfragment thereof, an HBV polymerase C-terminal domain comprising theamino acid sequence of SEQ ID NO: 182 or an immunogenic fragmentthereof, and an HBV Core protein comprising the amino acid sequence ofSEQ ID NO: 183 or an immunogenic fragment thereof. In some embodiments,the fusion protein comprises the amino acid sequence of SEQ ID NO: 175.

Also provided herein are fusion proteins comprising a herpes simplexvirus (HSV) glycoprotein (gD) sequence and the disclosed HBV Coreprotein, the HBV polymerase N-terminal domain, the HBV polymeraseC-terminal domain, or various combinations thereof.

The HSV gD is a receptor-binding glycoprotein of HSV. The gD ectodomainis organized in two structurally and functionally differentiatedregions: the amino-terminus, which includes the signal sequence andreceptor-binding sites; and the carboxy-terminus, which includes thepro-fusion domain and the transmembrane domain. gD interacts with theherpesvirus entry mediator (HVEM) receptor and the nectin receptors.Interaction of gD with the receptors results in the down-regulation ofthe HVEM receptors binding to BTLA or CD160, which are immunoinhibitorymolecules that are expressed on T cells. In some embodiments, thedisclosed fusion proteins comprising gD and the disclosed HBV Coreprotein, the HBV polymerase N-terminal domain, the HBV polymeraseC-terminal domain (referred to as “gDCore,” “gDPolN” or “gDPolC,”respectively), or combinations thereof are expected to enhance asubject's immune response against HBV to a greater extent compared tothe HBV Core and/or polymerase antigens alone (i.e. without gD).

Suitable HSV gD proteins for use in the disclosed fusion proteinsinclude wild-type or mutant gD that retains the ability to: 1) augmentstimulation of a CD8+ T cell response to an antigen; and/or 2) disruptan HVEM-BTLA pathway activity.

The fusion proteins can comprise the HBV Core protein or an immunogenicfragment thereof, HBV polymerase N-terminal domain or an immunogenicfragment thereof, HBV polymerase C-terminal domain or an immunogenicfragment thereof disclosed herein, or any combination thereof, anN-terminal HSV gD protein sequence, and a C-terminal HSV gD proteinsequence. The HBV Core protein, HBV polymerase N-terminal domain, andHBV polymerase C-terminal domain can be those provided in Table 9 or theimmunogenic fragments provided in Tables 3-5. The HBV Core protein, HBVpolymerase N-terminal domain, HBV polymerase C-terminal domain, orimmunogenic fragments thereof can be inserted between the N-terminal HSVgD protein sequence and the C-terminal HSV gD protein sequence. In someaspects, the N-terminal HSV gD protein sequence comprises the amino acidsequence of SEQ ID NO: 12 and the C-terminal HSV gD protein sequencecomprises the amino acid sequence of SEQ ID NO: 13. In some embodiments,the N-terminal HSV gD protein sequence comprises amino acid residues26-269 of SEQ ID NO: 12.

The fusion protein can comprise:

an N-terminal HSV gD sequence or a variant thereof;

an HBV Core protein comprising the amino acid sequence of SEQ ID NO: 6or an immunogenic fragment thereof; and

a C-terminal HSV gD sequence or a variant thereof.

The immunogenic fragment of the HBV Core protein can comprise any one ofSEQ ID NOs: 20-54, 180, or 183.

The fusion protein can comprise:

an N-terminal HSV gD sequence or a variant thereof;

an HBV Core protein comprising the amino acid sequence of SEQ ID NO: 180or SEQ ID NO: 183; and

a C-terminal HSV gD sequence or a variant thereof.

The fusion protein can comprise:

an N-terminal HSV gD sequence or a variant thereof;

an HBV polymerase N-terminal domain comprising the amino acid sequenceof SEQ ID NO: 8 or an immunogenic fragment thereof; and

a C-terminal HSV gD protein sequence or a variant thereof.

The immunogenic fragment of the HBV polymerase N-terminal domain cancomprise any one of SEQ ID NOs: 55-113, 178, or 181.

The fusion protein can comprise:

an N-terminal HSV gD sequence or a variant thereof;

an HBV polymerase N-terminal domain comprising the amino acid sequenceof SEQ ID NO: 178 or SEQ ID NO: 181; and

a C-terminal HSV gD protein sequence or a variant thereof.

The fusion protein can comprise:

an N-terminal HSV gD sequence or a variant thereof;

an HBV polymerase C-terminal domain comprising the amino acid sequenceof SEQ ID NO: 10 or an immunogenic fragment thereof; and

a C-terminal HSV gD protein sequence or a variant thereof.

The immunogenic fragment of the HBV polymerase C-terminal domain cancomprise any one of SEQ ID NOs: 114-172, 179, or 182.

The fusion protein can comprise:

an N-terminal HSV gD sequence or a variant thereof;

an HBV polymerase C-terminal domain comprising the amino acid sequenceof SEQ ID NO: 179 or SEQ ID NO: 182; and

a C-terminal HSV gD protein sequence or a variant thereof.

The fusion protein can comprise:

an N-terminal HSV gD sequence or a variant thereof;

an HBV sequence comprising:

-   -   (1) an HBV Core protein comprising the amino acid sequence of        SEQ ID NO: 6 or an immunogenic fragment thereof and an HBV        polymerase N-terminal domain comprising the amino acid sequence        of SEQ ID NO: 8 or an immunogenic fragment thereof;    -   (2) one or more immunogenic fragments of the HBV Core protein        comprising the amino acid sequence of SEQ ID NO: 6 and one or        more immunogenic fragments of the HBV polymerase N-terminal        domain comprising the amino acid sequence of SEQ ID NO: 8. For        example, one or more of SEQ ID NOs: 20-54 provided in Table 3        (immunogenic fragments of SEQ ID NO: 6) and one or more of SEQ        ID NOs: 55-113 provided in Table 4 (immunogenic fragments of SEQ        ID NO: 8);    -   (3) an HBV Core protein comprising the amino acid sequence of        SEQ ID NO: 6 or an immunogenic fragment thereof and an HBV        polymerase C-terminal domain comprising the amino acid sequence        of SEQ ID NO: 10 or an immunogenic fragment thereof;    -   (4) one or more immunogenic fragments of the HBV Core protein        comprising the amino acid sequence of SEQ ID NO: 6 and one or        more immunogenic fragments of the HBV polymerase C-terminal        domain comprising the amino acid sequence of SEQ ID NO: 10. For        example, one or more of SEQ ID NOs: 20-54 provided in Table 3        (immunogenic fragments of SEQ ID NO: 6) and one or more of SEQ        ID NOs: 114-172 provided in Table 5 (immunogenic fragments of        SEQ ID NO: 10);    -   (5) an HBV polymerase N-terminal domain comprising the amino        acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof        and an HBV polymerase C-terminal domain comprising the amino        acid sequence of SEQ ID NO: 10 or an immunogenic fragment        thereof;    -   (6) one or more immunogenic fragments of the HBV polymerase        N-terminal domain comprising the amino acid sequence of SEQ ID        NO: 8 and one or more immunogenic fragments of the HBV        polymerase C-terminal domain comprising the amino acid sequence        of SEQ ID NO: 10. For example, one or more of SEQ ID NOs: 55-113        provided in Table 4 (immunogenic fragments of SEQ ID NO: 8) and        one or more of SEQ ID NOs: 114-172 provided in Table 5        (immunogenic fragments of SEQ ID NO: 10);    -   (7) an HBV Core protein comprising the amino acid sequence of        SEQ ID NO: 6 or an immunogenic fragment thereof, an HBV        polymerase N-terminal domain comprising the amino acid sequence        of SEQ ID NO: 8 or an immunogenic fragment thereof, and an HBV        polymerase C-terminal domain comprising the amino acid sequence        of SEQ ID NO: 10 or an immunogenic fragment thereof; or    -   (8) one or more immunogenic fragments of the HBV Core protein        comprising the amino acid sequence of SEQ ID NO: 6, one or more        immunogenic fragments of the HBV polymerase N-terminal domain        comprising the amino acid sequence of SEQ ID NO: 8, and one or        more immunogenic fragments of the HBV polymerase C-terminal        domain comprising the amino acid sequence of SEQ ID NO: 10. For        example, one or more of SEQ ID NOs: 20-54 provided in Table 3        (immunogenic fragments of SEQ ID NO: 6), one or more of SEQ ID        NOs: 55-113 provided in Table 4 (immunogenic fragments of SEQ ID        NO: 8), and one or more of SEQ ID NOs: 114-172 provided in Table        5 (immunogenic fragments of SEQ ID NO: 10);    -   (9) an HBV polymerase N-terminal domain comprising the amino        acid sequence of SEQ ID NO: 178 or an immunogenic fragment        thereof, an HBV polymerase C-terminal domain comprising the        amino acid sequence of SEQ ID NO: 179 or an immunogenic fragment        thereof, and an HBV Core protein comprising the amino acid        sequence of SEQ ID NO: 180 or an immunogenic fragment thereof;        or    -   (10) an HBV polymerase N-terminal domain comprising the amino        acid sequence of SEQ ID NO: 181 or an immunogenic fragment        thereof, an HBV polymerase C-terminal domain comprising the        amino acid sequence of SEQ ID NO: 182 or an immunogenic fragment        thereof, and an HBV Core protein comprising the amino acid        sequence of SEQ ID NO: 183 or an immunogenic fragment thereof        and

a C-terminal HSV gD protein sequence or a variant thereof.

In some embodiments, the N-terminal HSV gD sequence can comprise atleast amino acids 1-269 of HSV gD. The N-terminal HSV gD sequence, forexample, can comprise the amino acid sequence of SEQ ID NO: 12. In someembodiments, the N-terminal HSV gD sequence comprises amino acidresidues 26-269 of SEQ ID NO: 12.

In some embodiments, the C-terminal HSV gD sequence comprises thetransmembrane domain of the HSV gD. The C-terminal HSV gD sequence, forexample, can comprise the amino acid sequence of SEQ ID NO: 13.

The fusion protein can comprise the amino acid sequence of SEQ ID NO: 14(corresponding to gDCore) or an immunogenic fragment thereof. The fusionprotein can comprise the amino acid sequence of SEQ ID NO: 16(corresponding to gDPolN) or an immunogenic fragment thereof. The fusionprotein can comprise the amino acid sequence of SEQ ID NO: 18(corresponding to gDPolC) or an immunogenic fragment thereof. In someembodiments, the amino acid sequence of any one of SEQ ID NOs: 14, 16,or 18, or the immunogenic fragment thereof, does not contain theN-terminal 25 amino acid signal peptide.

The fusion protein can comprise the amino acid sequence of SEQ ID NO:185 (gDHBV2). The fusion protein can comprise the amino acid sequence ofSEQ ID NO: 187 (gDHBV3).

Nucleic acid molecules encoding any of the disclosed fusion proteins arealso provided. In some embodiments, the nucleic acid molecule comprisesthe nucleotide sequence of SEQ ID NO: 15 (corresponding to gDCore). Insome embodiments, the nucleic acid molecule comprises the nucleotidesequence of SEQ ID NO: 17 (corresponding to gDPolN). In someembodiments, the nucleic acid molecule comprises the nucleotide sequenceof SEQ ID NO: 19 (corresponding to gDPolC).

The nucleic acid molecule can comprise the nucleotide sequence of SEQ IDNO: 184 (gDHBV2). The nucleic acid molecule can comprise the nucleotidesequence of SEQ ID NO: 186 (gDHBV3).

Vectors comprising the nucleic acid molecules encoding the fusionproteins are also disclosed. Suitable vectors include those describedabove including, for example, an adenoviral vector. In some embodiments,the adenoviral vector is an AdC6 vector. In some embodiments, theadenoviral vector is an AdC7 vector. The vector can comprise thenucleotide sequence of SEQ ID NO: 184 (gDHBV2). In some aspects, thevector is an AdC6 vector that comprises the nucleotide sequence of SEQID NO: 184 (gDHBV2). In some aspects, the vector is an AdC7 vector thatcomprises the nucleotide sequence of SEQ ID NO: 184 (gDHBV2). The vectorcan comprise the nucleotide sequence of SEQ ID NO: 186 (gDHBV3). In someaspects, the vector is an AdC6 vector that comprises the nucleotidesequence of SEQ ID NO: 186 (gDHBV3). In some aspects, the vector is anAdC7 vector that comprises the nucleotide sequence of SEQ ID NO: 186(gDHBV3).

Vaccines comprising any of the disclosed vectors are also provided. Thevaccine can further comprise a pharmaceutically acceptable carrier orpharmaceutical acceptable excipient as disclosed above. The vaccine cancomprise a vector comprising the nucleotide sequence of SEQ ID NO: 184(gDHBV2). In some aspects, the vaccine comprises an AdC6 vector thatcomprises the nucleotide sequence of SEQ ID NO: 184 (gDHBV2). In someaspects, the vaccine comprises an AdC7 vector that comprises thenucleotide sequence of SEQ ID NO: 184 (gDHBV2). The vaccine can comprisea vector that comprises the nucleotide sequence of SEQ ID NO: 186(gDHBV3). In some aspects, the vaccine comprises an AdC6 vector thatcomprises the nucleotide sequence of SEQ ID NO: 186 (gDHBV3). In someaspects, the vaccine comprises an AdC7 vector that comprises thenucleotide sequence of SEQ ID NO: 186 (gDHBV3).

Provided herein are methods of inducing an immune response to HBV in asubject, the methods comprising providing to the subject an effectiveamount of any of the disclosed fusion proteins, any of the disclosednucleic acid molecules, any of the disclosed vectors, or any of thedisclosed vaccines to thereby induce an immune response to HBV. In someembodiments, the methods comprise providing to the subject an effectiveamount of any of the disclosed fusion proteins to thereby induce animmune response to HBV. In some embodiments, the methods compriseproviding to the subject an effective amount of any of the disclosednucleic acid molecules to thereby induce an immune response to HBV. Insome embodiments, the methods comprise providing to the subject aneffective amount of any of the disclosed vectors to thereby induce animmune response to HBV. In some embodiments, the methods compriseproviding to the subject an effective amount of any of the disclosedvaccines to thereby induce an immune response to HBV.

The methods can comprise providing to the subject an effective amount ofa vaccine comprising an AdC6 vector, wherein the AdC6 vector comprises afusion protein comprising the amino acid sequence of any one of SEQ IDNOs: 14, 16, or 18, or an immunogenic fragment thereof. In someembodiments, the methods further comprise providing to the subject,subsequent to providing the vaccine comprising the AdC6 vector, avaccine comprising an AdC7 vector comprising a fusion protein comprisingthe amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18, or animmunogenic fragment thereof. Such prime-boost methods can comprise:

-   -   Providing to the subject a vaccine comprising an AdC6 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 14, or an immunogenic fragment thereof, and        subsequently providing to the subject a vaccine comprising an        AdC7 vector comprising a fusion protein comprising the amino        acid sequence of SEQ ID NO: 14, or an immunogenic fragment        thereof. In some embodiments, the amino acid sequence of SEQ ID        NO: 14, or an immunogenic fragment thereof, does not contain the        N-terminal 25 amino acid signal peptide;    -   Providing to the subject a vaccine comprising an AdC6 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 16, or an immunogenic fragment thereof, and        subsequently providing to the subject a vaccine comprising an        AdC7 vector comprising a fusion protein comprising the amino        acid sequence of SEQ ID NO: 16, or an immunogenic fragment        thereof. In some embodiments, the amino acid sequence of SEQ ID        NO: 16, or an immunogenic fragment thereof, does not contain the        N-terminal 25 amino acid signal peptide; or    -   Providing to the subject a vaccine comprising an AdC6 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 18, or an immunogenic fragment thereof, and        subsequently providing to the subject a vaccine comprising an        AdC7 vector comprising a fusion protein comprising the amino        acid sequence of SEQ ID NO: 18, or an immunogenic fragment        thereof. In some embodiments, the amino acid sequence of SEQ ID        NO: 18, or an immunogenic fragment thereof, does not contain the        N-terminal 25 amino acid signal peptide.

The methods can comprise providing to the subject an effective amount ofa vaccine comprising an AdC7 vector, wherein the AdC7 vector comprises afusion protein comprising the amino acid sequence of any one of SEQ IDNOs: 14, 16, or 18, or an immunogenic fragment thereof. In someembodiments, the methods further comprise providing to the subject,subsequent to providing the vaccine comprising the AdC7 vector, avaccine comprising an AdC6 vector comprising a fusion protein comprisingthe amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18, or animmunogenic fragment thereof. Such prime-boost methods can comprise:

-   -   Providing to the subject a vaccine comprising an AdC7 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 14, or an immunogenic fragment thereof, and        subsequently providing to the subject a vaccine comprising an        AdC6 vector comprising a fusion protein comprising the amino        acid sequence of SEQ ID NO: 14, or an immunogenic fragment        thereof. In some embodiments, the amino acid sequence of SEQ ID        NO: 14, or an immunogenic fragment thereof, does not contain the        N-terminal 25 amino acid signal peptide;    -   Providing to the subject a vaccine comprising an AdC7 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 16, or an immunogenic fragment thereof, and        subsequently providing to the subject a vaccine comprising an        AdC6 vector comprising a fusion protein comprising the amino        acid sequence of SEQ ID NO: 16, or an immunogenic fragment        thereof. In some embodiments, the amino acid sequence of SEQ ID        NO: 16, or an immunogenic fragment thereof, does not contain the        N-terminal 25 amino acid signal peptide; or    -   Providing to the subject a vaccine comprising an AdC7 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 18, or an immunogenic fragment thereof, and        subsequently providing to the subject a vaccine comprising an        AdC6 vector comprising a fusion protein comprising the amino        acid sequence of SEQ ID NO: 18, or an immunogenic fragment        thereof. In some embodiments, the amino acid sequence of SEQ ID        NO: 18, or an immunogenic fragment thereof, does not contain the        N-terminal 25 amino acid signal peptide.

The methods can comprise providing to the subject an effective amount ofa vaccine comprising an AdC6 vector, wherein the AdC6 vector comprises afusion protein comprising the amino acid sequence of SEQ ID NO: 185 or187, or an immunogenic fragment thereof. In some embodiments, themethods further comprise providing to the subject, subsequent toproviding the vaccine comprising the AdC6 vector, a vaccine comprisingan AdC7 vector comprising a fusion protein comprising the amino acidsequence of SEQ ID NO: 185 or 187, or an immunogenic fragment thereof.Such prime-boost methods can comprise:

-   -   Providing to the subject a vaccine comprising an AdC6 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 185, or an immunogenic fragment thereof, and        subsequently providing to the subject a vaccine comprising an        AdC7 vector comprising a fusion protein comprising the amino        acid sequence of SEQ ID NO: 185, or an immunogenic fragment        thereof. In some embodiments, the amino acid sequence of SEQ ID        NO: 185, or an immunogenic fragment thereof, does not contain        the N-terminal 25 amino acid signal peptide; or    -   Providing to the subject a vaccine comprising an AdC6 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 187, or an immunogenic fragment thereof, and        subsequently providing to the subject a vaccine comprising an        AdC7 vector comprising a fusion protein comprising the amino        acid sequence of SEQ ID NO: 187, or an immunogenic fragment        thereof. In some embodiments, the amino acid sequence of SEQ ID        NO: 187, or an immunogenic fragment thereof, does not contain        the N-terminal 25 amino acid signal peptide.

The methods can comprise providing to the subject an effective amount ofa vaccine comprising an AdC7 vector, wherein the AdC7 vector comprises afusion protein comprising the amino acid sequence of SEQ ID NO: 185 or187, or an immunogenic fragment thereof. In some embodiments, themethods further comprise providing to the subject, subsequent toproviding the vaccine comprising the AdC7 vector, a vaccine comprisingan AdC6 vector comprising a fusion protein comprising the amino acidsequence of SEQ ID NO: 185 or 187, or an immunogenic fragment thereof.Such prime-boost methods can comprise:

-   -   Providing to the subject a vaccine comprising an AdC7 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 185, or an immunogenic fragment thereof, and        subsequently providing to the subject a vaccine comprising an        AdC6 vector comprising a fusion protein comprising the amino        acid sequence of SEQ ID NO: 185, or an immunogenic fragment        thereof. In some embodiments, the amino acid sequence of SEQ ID        NO: 185, or an immunogenic fragment thereof, does not contain        the N-terminal 25 amino acid signal peptide; or    -   Providing to the subject a vaccine comprising an AdC7 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 187, or an immunogenic fragment thereof, and        subsequently providing to the subject a vaccine comprising an        AdC6 vector comprising a fusion protein comprising the amino        acid sequence of SEQ ID NO: 187, or an immunogenic fragment        thereof. In some embodiments, the amino acid sequence of SEQ ID        NO: 187, or an immunogenic fragment thereof, does not contain        the N-terminal 25 amino acid signal peptide.

The immune response induced by the disclosed methods include, but is notlimited to, T cell responses, B cell responses, or both (i.e. cellularand/or humoral immune responses). The immune response can be a primaryimmune response or a secondary immune response. The disclosed methodscan induce a subject's immune response against HBV to a greater extentcompared to the HBV Core or polymerase antigens alone (i.e. without gD).

The disclosed methods can be used for both therapeutic treatment andprophylactic or preventative measures and can reduce the severity and/orfrequency of symptoms, eliminate symptoms and/or the underlying cause ofthe symptoms, reduce the frequency or likelihood of symptoms and/ortheir underlying cause, and improve or remediate damage caused, directlyor indirectly, by HBV. Treatment also includes prolonging survival ascompared to the expected survival of a subject not receiving treatment.Subjects to be treated include those that have HBV as well as thoseprone to have HBV or those in which HBV is to be prevented.

The amount of the disclosed fusion proteins, nucleic acid molecules,vectors, or vaccines needed to thereby induce an immune response to HBV(e.g. a “effective amount”) may vary according to factors such as thedisease state, age, sex, and weight of the subject, and the ability ofthe fusion proteins, nucleic acid molecules, vectors, or vaccines tocause a desired response in the subject. Exemplary indicators of aneffective amount include, for example, improved well-being of thesubject and reduction, elimination, or prevention of HBV symptoms.

Also provided is the use of any of the disclosed fusion proteins,nucleic acid molecules, vectors, or vaccines in the manufacture of amedicament for inducing an immune response to HBV in a subject.

The disclosed fusion proteins, nucleic acid molecules, vectors, orvaccines for use in inducing an immune response to HBV in a subject isalso provided.

EXAMPLES

The following examples are provided to further describe some of theembodiments disclosed herein. The examples are intended to illustrate,not to limit, the disclosed embodiments.

Generation of an Epitope-Optimized Core Sequence

Hepatitis B virus (HBV) can be grouped into several genotypes, based onphylogenic clustering. To assist in the development of an antigen insertfor a multi-genotype HBV vaccine for patients with chronic infections, apreliminary bioinformatics evaluation of the genes encoding the HBV Coreand HBV polymerase across genotypes A, B, C and D was conducted.

The Core amino acid sequences from the four major HBV clades weredownloaded as aligned ClustalW sequences from Hepatitis B Virus database(HBVdb) (release version 45.0; last updated on Aug. 2, 2018). The aminoacid sequences represented thousands of HBV genomes inputted from usersacross Europe, as summarized in the following table.

TABLE 1 Number of unique Core genomes analyzed Genotype HBV Gene UniqueGenomes Analyzed HBV genotype A Core 1,482 HBV genotype B 2,800 HBVgenotype C 2,768 HBV genotype D 1,579

“Consensus” Core sequences were first identified for each genotype usingthe Shannon Entropy tool hosted by the Los Alamos National Laboratory(www.hiv.lanl.gov/content/sequence/ENTROPY/entropy), which calculatedthe variation and frequency at each amino acid position. Thesecalculations were repeated for each genotype, generating four“consensus” Core sequences, one for each genotype analyzed (SEQ ID NOs:1-4):

Genotype A Consensus (SEQ ID NO: 1) MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLAT WVGNNLeDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPIL STLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC- Genotype B Consensus (SEQ ID NO: 2) MDID p YKEFGAS vELLSFLPSDFFPS i RDLLDTA s ALYREALESPEHCSPHHTALRQAI l CWGELMNLAT WVGSNL eDPASRELVV s YVNVNMGLK i RQLLWFHI SCLTFGRETVLEYLVSFGVWIRTP p AYRP p NAPILSTLPETTVVRRRGRSPRRRIPSPRRRRSQSPRRRR SQSRE s QC- Genotype C Consensus(SEQ ID NO: 3) MDID p YKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLAT WVGSNLEDPASRELVV s YVNVNMGLK iRQlLWFHI SCLTFGRETVLEYLVSFGVWIRTP p AYRPPNAPILSTLPETTVVRRRGRSPRRRIPSPRRRRSQSPRRRS QSRESQC - Genotype D Consensus(SEQ ID NO: 4) MDIDPYKEFGA t VELLSFLP s DFFPSVRDLLDTAS ALYR eALESPEHCSPHHTALRQAILCWG e LMtLAT WVG g NLEDP a SRDLVVSYVNTN mGLKFRQLLWFHI SCLTFGR e TV i EYLVSFGVWIRTP p AYRPPNAPIL STLPETTV vRRRGRSPRRRIPSPRRRTSQSPRRRR SQSRESQC-(Bold, underlined residues represent aminoacids having less than 90% frequency).

The above “consensus” Core sequences were combined to generate anepitope-optimized Core sequence. Conserved amino acids were identifiedat each amino acid residue of the Core protein from each genotype (A, B,C and D) and the frequency and variation within a given sample ofgenotype genomes was determined. To select amino acids at sites ofvariation, each variation was tested using epitope prediction algorithmsacross multiple HLA types and the most immunogenic sequence wasselected. Specifically:

-   -   (1) Each residue across the four genotypes that was identical        were maintained. The genome weighted frequency was also        calculated to inform the variability with spacer added, where        applicable, to align the sequences for diversity.    -   (2) Residues that were not identical across the four genotypes        were identified and the amino acid diversity was recorded (see        Table 2). The initial Core sequence (SEQ ID NO: 5) is provided        below, with the residues that were not identical across the four        genotypes labeled as X₁-X₁₁ and the residues having less than        90% frequency in bold, underlined font:

MDID P YKEFGAX ₁VELLSFLPSDFFPSX ₂DLLDTASALYREALESPEHCSPHHTALRQAILCWGELMX ₃LATWVGX ₄NLeDPASRX ₅L VVX ₆YVNX ₇NMGLKX₈RQLLWFHISCLTFGRETVX ₉EYLVSFGV WIRTP P AYRP P NAPILSTLPETTVVRRRX ₁₀ X₁₁GRSPRRRITS PRRRRSQSPRRRRSQSRESQC

TABLE 2 Residues that were not identical across the four genotypesResidue # X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ X₁₁ Genotype A T V T N D N T IL D R Consensus A - Consensus 95.4% 98.2% 96.0% 94.0% 99.5% 98.9% 98.3%98.8% 98.2% 95.8% 99.5% Frequency Genotype B S i N S E s V i L — —Consensus B - Consensus 97.1% 89.7% 93.5% 90.1% 91.5% 75.2% 93.6% 80.1%99.4% — — Frequency Genotype C S I N S E s V i L — — Consensus C -Consensus 99.4% 90.3% 97.3% 96.3% 97.0% 83.7% 97.8% 78.7% 98.8% — —Frequency Genotype D t V t g D S T F i — — Consensus D - Consensus 75.7%97.0% 87.5% 58.7% 98.4% 93.7% 96.5% 99.1% 77.8% — — Frequency

-   -   (3) To determine the final amino acid at these positions,        epitope prediction algorithms were used to select the        appropriate amino acid. For amino acids that showed variability        between the genotypes, amino acids that were present in 3 of the        genotypes were selected or an MHC class I epitope prediction        software was used to select the most immunogenic amino acids.        This approach maximized the potential immunogenicity across the        greatest number of HLA types. The epitope-optimized Core        sequence across all genotypes and within genotypes is shown        below (SEQ ID NO: 6):

DIDPYKEFGATVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRR SQSRESQC

The average variation at each site across all genomes, weighted by thenumber of clade-specific genomes analyzed, was calculated and showedareas and residues of higher and greater conservation. FIG. 1.

Generation of Epitope-Optimized Polymerase Sequences

An epitope-optimized polymerase sequence was generated from the fourmajor HBV clades as discussed above for the Core sequence. Because thepolymerase is long, two fragments—an N-terminal fragment (from which ahighly variable segment between the genotypes was removed) and aC-terminal fragment—were generated. Both fragments are approximately 300amino acids in length. The epitope-optimized polymerase amino acidsequences are shown below and in Table 9:

Epitope-optimized HBV polymerase N-terminal amino acid sequence(SEQ ID NO: 8): PLSYQHFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGITRVSWPKFAVPNLQSLTNLLS SNLSWLSLDVSAAFYHIPLHPAAMPEpitope-optimized HBV polymerase C-terminal amino acid sequence(SEQ ID NO: 10): HLLVGSSGLSRYVARLSSNSRIINHQHGTMQNLHDSCSRNLYVSLLLLYKTFGRKLHLYSHPIILKTKRWGYSLNFMGYVIGSWGSLPQDHIIQKIKECFRKLPVNRPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQSKQAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQRMRGTFVAPLP1HTAELLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDPSRGRLGLSR PLLRLPFRPTTGRTSLYAVSPSV

Generation of AdC6 and AdC7 Vectors Expressing the Epitope-OptimizedCore and Polymerase Sequences

The genes encoding the epitope-optimized Core or polymerase amino acidsequences were cloned into transfer vectors that contained the herpessimplex virus (HSV) glycoprotein D (gD) sequence under the control ofthe CMV promoter. The genes were then cloned into the El-deleted, E3 ORF3, 4, 5, 6, and 7-deleted replication deficient adenoviral vector (asdescribed in PCT/US2017/043315) to generate the following vectors:

-   -   AdC6 containing the epitope-optimized Core sequence fused to gD        (AdC6-gDCore);    -   AdC6 containing the epitope-optimized polymerase N-terminal        sequence fused to gD (AdC6-gDPolN);    -   AdC6 containing the epitope-optimized polymerase C-terminal        sequence fused to gD (AdC6-gDPolC);    -   AdC7 containing the epitope-optimized Core sequence fused to gD        (AdC7-gDCore);    -   AdC7 containing the epitope-optimized polymerase N-terminal        sequence fused to gD (AdC7-gDPolN); and    -   AdC7 containing the epitope-optimized polymerase C-terminal        sequence fused to gD (AdC7-gDPolC).

Correct clones were identified by restriction enzyme digest and thecloning sites were sequenced. Vectors were rescued and expanded in HEK293 cells, purified by cesium chloride (CsCl) gradient centrifugation,and the vector concentration (vp) was determined by spectrophotometry.Vectors were titrated for infectious units upon their expansion inserial dilutions in HEK 293 cells, followed by isolation and reversetranscription of RNA and a nested hexon-specific PCR reaction. Geneticintegrity of the vectors was determined by restriction enzyme digestfollowed by gel electrophoresis of purified viral DNA. Proteinexpression was determined by Western blotting using gD-specificantibodies. Genetic stability was determined by serial passages (12-15)of the vectors in HEK 293 cells followed by restriction enzyme digest ofpurified viral DNA and gel electrophoresis.

Testing of Immunogenicity of Vaccines in Mice

C57Bl/6, BALB/c, and HLA-A2 tg mice (n=5 per group) were injected withvarious concentrations of each of the above vectors. Naïve mice servedas controls. Mice were bled at different times after the injection andfrequencies of insert-specific CD8+ and CD4+ T cells were determined byintracellular cytokine staining (ICS) for IFN-γ. Two months after thefirst injection, AdC6-immune mice were boosted with the heterologousvector (AdC7) expressing the same insert. Frequencies of HBV-specific Tcells were tested again. Results after priming are shown in FIG. 2A-2Fand FIG. 3A (C57Bl/6 mice), FIG. 3B (BALB/c mice) and FIG. 3C (HLA-A2mice). Results after the boost are shown in FIGS. 4A-4C and 5A-5B.

C57Bl/6 mice showed a very robust CD8+ T cell response to theepitope-optimized polymerase N-terminal sequence and lower responses tothe epitope-optimized polymerase C-terminal sequence and theepitope-optimized Core sequence, while CD4+ responses were betteragainst the epitope-optimized Core sequence and the epitope-optimizedpolymerase C-terminal sequence (FIG. 2A-FIG. 2F). Epitope mapping inC57Bl/6 mice showed higher and broader responses to PolN than PolC (FIG.3A). Within PolN a total of 14 peptides were recognized by CD8+ T cellswhile within PolC only two adjacent peptides, which most likely reflectone epitope, were recognized. CD4+ T cells failed to respond to PolN orPolC. This pattern was largely mirrored in BALB/c mice, where CD8+ Tcell responses were highest against PolN with recognition of 12 peptidesfollowed by PolC with recognition of 4 peptides (FIG. 3B). Responses toCore were low but surprisingly broad with recognition of 10 peptides(FIG. 3B). BALB/c CD4+ T cells responded best to Core with recognitionof 15 peptides with lower recognition of PolC (4 peptides) or PolN (2peptides). CD8+ T cell responses were also tested in HLA-A2 tg micewhere PolN again triggered the highest response involving 12 peptides(FIG. 3C). The response to PolC was lower but broader (16 peptides)while only one peptide of Core was detected (FIG. 3C). The sequences ofthe peptides tested in the priming experiments are provided in Table 3(Core peptides), Table 4 (PolN peptides), and Table 5 (PolC peptides).The peptide composition of the peptide pools from the primingexperiments are provided in Tables 6-8. Overall these data show that theinserts elicited detectable T cell responses that in most cases weredirected against multiple epitopes within each sequence.

TABLE 3 Epitope-Optimized Core Peptides SEQ Amino Acid ID PeptideSequence NO: Prime 1 DIDPYKEFGATVELL 20 CD8 (B/c) 2 KEFGATVELLSFLPS 21CD8 (B/c) 3 TVELLSFLPSDFFPS 22 CD8 (B/c) 4 SFLPSDFFPSIRDLL 23 CD8 (B/c)5 DFFPSIRDLLDTASA 24 CD8 (B/c) 6 IRDLLDTASALYREA 25 7 DTASALYREALESPE 26CD4 (B/c) 8 LYREALESPEHCSPH 27 CD4 (Bl/6); CD4 (B/c) 9 LESPEHCSPHHTALR28 CD4 (B/c) 10 HCSPHHTALRQAILC 29 CD4 (B/c) 11 HTALRQAILCWGELM 30CD4 (B/c) 12 QAILCWGELMTLATW 31 13 WGELMTLATWVGSNL 32 CD8 (B/c);CD4 (B/c); CD8 (HLA) 14 TLATWVGSNLEDPAS 33 CD8 (B/c); CD4 (B/c) 15VGSNLEDPASRELVV 34 CD8 (B/c); CD4 (B/c) 16 EDPASRELVVSYVNV 35 CD8 (B/c);CD4 (B/c) 17 RELVVSYVNVNMGLK 36 CD8 (B/c); CD4 (B/c) 18 SYVNVNMGLKIRQLL37 19 NMGLKIRQLLWFHIS 38 CD4 (B/c) 20 IRQLLWFHISCLTFG 39 CD4 (B/c) 21WFHISCLTFGRETVI 40 CD4 (B/c) 22 CLTFGRETVIEYLVS 41 CD4 (B/c) 23RETVIEYLVSFGVWI 42 CD4 (B/c) 24 EYLVSFGVWIRTPPA 43 25 FGVWIRTPPAYRPPN 4426 RTPPAYRPPNAPILS 45 CD8 (Bl/6) 27 YRPPNAPILSTLPET 46 28APILSTLPETTVVRR 47 CD4 (Bl/6) 29 TLPETTVVRRRDRGR 48 CD8 (Bl/6) 30TVVRRRDRGRSPRRR 49 31 RDRGRSPRRRTPSPR 50 32 SPRRRTPSPRRRRSQ 51 33TPSPRRRRSQSPRRR 52 34 RRRSQSPRRRRRSQSR 53 35 SPRRRRRSQSRESQC 54 B/c= BALB/c; Bl/6 = C57Bl/6; HLA = HLA-A2

TABLE 4 Epitope-Optimized PoIN Peptides Amino Acid SEQ ID Prime PeptideSequence NO: 1 PLSYQHFRKLLLLDE 55 2 HFRKLLLLDEEAGPL 56 3 LLLDEEAGPLEEELP57 4 EAGPLEEELPRLADE 58 5 EEELPRLADEGLNRR 59 6 RLADEGLNRRVAEDL 60 7GLNRRVAEDLNLGNL 61 8 VAEDLNLGNLNVSIP 62 9 NLGNLNVSIPWTHKV 63 10NVSIPWTHKVGNFTG 64 CD4 (B/c) 11 WTHKVGNFTGLYSST 65 12 GNFTGLYSSTVPVFN 6613 LYS STVPVFNPEWQT 67 14 VPVFNPEWQTPSFPK 68 CD4 (B/c) 15PEWQTPSFPKIHKLQE 69 16 PSFPKIHKLQEDIVDR 70 17 IHKLQEDIVDRCKQFV 71 18EDIVDRCKQFVGPLTV 72 19 RCKQFVGPLTVNEKRR 73 20 VGPLTVNEKRRLKLIM 74 21VNEKRRLKLIMPARFY 75 22 RLKLIMPARFYPNVTK 76 23 MPARFYPNVTKYLPLD 77 24YPNVTKYLPLDKGIKP 78 25 KYLPLDKGIKPYYPEH 79 26 DKGIKPYYPEHAVNHY 80 27PYYPEHAVNHYFQTRH 81 28 HAVNHYFQTRHYLHTL 82 29 YFQTRHYLHTLWKAGI 83 30HYLHTLWKAGILYKRE 84 31 LWKAGILYKRETTRSA 85 32 ILYKRETTRSASFCGS 86 33ETTRSASFCGSPYSWE 87 34 ASFCGSPYSWEQELQH 88 CD8 (Bl/6); CD8 (B/c);CD8 (HLA) 35 SPYSWEQELQHGSCWW 89 CD8 (Bl/6); CD8 (B/c); CD8 (HLA) 36EQELQHGSCWWLQFRN 90 37 HGSCWWLQFRNSKPCS 91 CD8 (Bl/6); CD8 (B/c);CD8 (HLA) 38 WLQFRNSKPCSEYCLT 92 CD8 (Bl/6); CD8 (B/c); CD8 (HLA) 39NSKPCSEYCLTHLVNL 93 CD8 (Bl/6) 40 SEYCLTHLVNLLEDWG 94 CD8 (Bl/6);CD8 (B/c); CD8 (HLA) 41 THLVNLLEDWGPCDEH 95 42 LLEDWGPCDEHGEHHI 96 43GPCDEHGEHHIRIPRT 97 44 HGEHHIRIPRTPARVT 98 45 IRIPRTPARVTGGVFL 99 46TPARVTGGVFLVDKNP 100 47 TGGVFLVDKNPHNTAE 101 48 LVDKNPHNTAESRLVV 102 49PHNTAESRLVVDFSQF 103 50 ESRLVVDFSQFSRGIT 104 CD8 (Bl/6); CD8 (B/c);CD8 (HLA) 51 VDFSQFSRGITRVSWP 105 CD8 (Bl/6); CD8 (B/c); CD8 (HLA) 52FSRGITRVSWPKFAVP 106 53 TRVSWPKFAVPNLQSL 107 CD8 (Bl/6); CD8 (B/c);CD8 HLA) 54 PKFAVPNLQSLTNLLS 108 CD8 (Bl/6); CD8 (B/c); CD8 (HLA) 55PNLQSLTNLLSSNLSW 109 CD8 (Bl/6) 56 LTNLLSSNLSWLSLDV 110 CD8 (Bl/6);CD8 (B/c); CD8 (HLA) 57 SSNLSWLSLDVSAAFY 111 58 WLSLDVSAAFYHIPLH 112CD8 (Bl/6); CD8 (B/c); CD8 (HLA) 59 VSAAFYHIPLHPAAMP 113 CD8 (Bl/6);CD8 (B/c); CD8 (HLA) B/c = BALB/c; Bl/6 = C57Bl/6; HLA = HLA-A2

TABLE 5 Epitope-Optimized PoIC Peptides Peptide Amino Acid SEQ PrimeSequence ID NO: 1 HLLVGSSGLSRYVAR 114 2 SSGLSRYVARLSSNSR 115 3RYVARLSSNSRIINHQ 116 4 LSSNSRIINHQHGTMQ 117 5 RIINHQHGTMQNLHDS 118 6QHGTMQNLHDSCSRNL 119 7 QNLHDSCSRNLYVSLL 120 8 SCSRNLYVSLLLLYKT 121 9LYVSLLLLYKTFGRKL 122 10 LLLYKTFGRKLHLYSH 123 CD8 (HLA) 11TFGRKLHLYSHPIILK 124 12 LHLYSHPIILKTKRWG 125 CD8 (B/c); CD8 (HLA) 13HPIILKTKRWGYSLNF 126 CD8 (HLA) 14 KTKRWGYSLNFMGYVI 127 15GYSLNFMGYVIGSWGS 128 CD8 (HLA) 16 FMGYVIGSWGSLPQDH 129 17IGSWGSLPQDHIIQKI 130 18 SLPQDHIIQKIKECFR 131 19 HIIQKIKECFRKLPVN 132 20IKECFRKLPVNRPIDW 133 21 RKLPVNRPIDWKVCQR 134 22 NRPIDWKVCQRIVGLL 135 23WKVCQRIVGLLGFAAP 136 24 RIVGLLGFAAPFTQCG 137 25 LGFAAPFTQCGYPALM 138 26PFTQCGYPALMPLYAC 139 CD8 (HLA) 27 GYPALMPLYACIQSKQ 140 28MPLYACIQSKQAFTFS 141 CD8 (B/c); CD8 (HLA) 29 CIQSKQAFTFSPTYKA 142CD8 (HLA) 30 QAFTFSPTYKAFLSKQ 143 31 SPTYKAFLSKQYLNLY 144 CD8 (Bl/6);CD8 (HLA) 32 AFLSKQYLNLYPVARQ 145 CD8 (Bl/6) 33 QYLNLYPVARQRPGLC 146 34YPVARQRPGLCQVFAD 147 CD8 (HLA) 35 QRPGLCQVFADATPTG 148 CD4 (B/c) 36CQVFADATPTGWGLAM 149 CD8 (B/c); CD8 (HLA) 37 DATPTGWGLAMGHQRM 150CD8 (HLA) 38 GWGLAMGHQRMRGTFV 151 39 MGHQRMRGTFVAPLPI 152 CD4 (B/c);CD8 (HLA) 40 MRGTFVAPLPIHTAEL 153 41 VAPLPIHTAELLAACF 154 42IHTAELLAACFARSRS 155 CD8 (HLA) 43 LLAACFARSRSGAKIL 156 44FARSRSGAKILGTDNS 157 CD8 (B/c); CD8 (HLA) 45 SGAKILGTDNSVVLSR 158CD8 (HLA) 46 LGTDNSVVLSRKYTSF 159 47 SVVLSRKYTSFPWLLG 160 48RKYTSFPWLLGCAANW 161 CD8 (HLA) 49 FPWLLGCAANWILRGT 162 50GCAANWILRGTSFVYV 163 51 WILRGTSFVYVPSALN 164 CD4 (B/c) 52TSFVYVPSALNPADDP 165 53 VPSALNPADDPSRGRL 166 54 NPADDPSRGRLGLSRP 167 55PSRGRLGLSRPLLRLP 168 CD4 (B/c) 56 LGLSRPLLRLPFRPTT 169 57PLLRLPFRPTTGRTSL 170 58 PFRPTTGRTSLYAVSP 171 59 TGRTSLYAVSPSV 172 B/c= BALB/c; Bl/6 = C57Bl/6; HLA = HLA-A2

TABLE 6 Epitope-Optimized Core Pool Core Matrix A B C D E F G 1 2 3 4 56 H 7 8 9 10 11 12 I 13 14 15 16 17 18 J 19 20 21 22 23 24 K 25 26 27 2829 30 L 31 32 33 34 35

TABLE 7 Epitope-Optimized PolN Pool Pol N Matrix A B C D E F G H I 1 2 34 5 6 7 8 J 9 10 11 12 13 14 15 16 K 17 18 19 20 21 22 23 24 L 25 26 2728 29 30 31 32 M 33 34 35 36 37 38 39 40 N 41 42 43 44 45 46 47 48 O 4950 51 52 53 54 55 56 P 57 58 59

TABLE 8 Epitope-Optimized PolC Pool Pol C Matrix A B C D E F G H I 1 2 34 5 6 7 8 J 9 10 11 12 13 14 15 16 K 17 18 19 20 21 22 23 24 L 25 26 2728 29 30 31 32 M 33 34 35 36 37 38 39 40 N 41 42 43 44 45 46 47 48 O 4950 51 52 53 54 55 56 P 57 58

After the boost, which was tested in C57Bl/6, BALB/c and HLA-A2 tg mice,increases in responses were mainly seen for inserts and at vector dosesthat upon priming induced suboptimal responses, i.e., for Core tested atthe 1×10⁹ vp vector dose (FIG. 4A-4C). Although booster immunizationfailed to increase the response to PolN or PolC when vectors wereinjected at high doses, the boost nevertheless broadened the T cellresponses (FIG. 5A-5C)

Immunogenicity Summary

The above results illustrate that:

-   -   The vaccines are immunogenic: PolN>PolC>Core for CD8+ T cells;        Core>PolC>PolN for CD4+ T cell responses;    -   Immune responses can be boosted by a heterologous vaccine        carrier;    -   Immune responses are broad; and    -   The breadth of the T cell responses increases after the boost.

Effect of Vaccination on HBV Titers Low Dose AAV-1.3HBV Challenge

A group of 3 mice were challenged with 1×10¹⁰, 1×10¹¹ or 1.5×10¹¹ vg ofAAV-1.3HBV and were vaccinated with AdC6-gDPolN 8 weeks later. Viraltiters were tested 8 weeks after vaccination and compared topre-vaccination titers. FIG. 6 shows viral changes from baseline foreach treatment group.

Epitope Shifting

CD8+ T cells to HBV antigens become exhausted during chronic HBVinfections. Progression towards exhaustion is more rapid and pronouncedfor CD8+ T cells to dominant, as compared to subdominant, epitopes. Theunderlying reason is that exhaustion is driven by overwhelmingantigen-driven stimulation through the T cell receptor; dominantepitopes are presented at higher levels on MHC class I antigensexpressed by antigen presenting cells than subdominant epitopes withlower avidity to their restricting elements. Typical vaccine approachesprimarily induce immune responses to dominant epitopes. Therapeuticvaccines should take into account loss of T cells to dominant epitopesduring chronic virus infections and should be designed to favorexpansion of CD8+ T cells to subdominant epitopes, which have a higherlikelihood of resisting disease-driven exhaustion, translating tosuperior disease control.

The epitope profile in naïve mice immunized with an adenovirus vectorcomprising a nucleic acid sequence encoding the HBV polymeraseN-terminal domain (PolN) fused to the herpes simplex virus glycoproteinD (“AdC6-gDPolN”, wherein the amino acid sequence of gDPolN is SEQ IDNO: 16) was determined. Responses in mice that had not been pre-treatedwith the AAV8-1.3HBV vector were compared to those obtained in miceinfected with an AAV8 vector expressing the 1.3HBV genome prior tovaccination with the AdC6-gDPolN. The AAV8-1.3HBV vector induced hightiters of HBV in serum, which could drive CD8+ T cell exhaustion.

In the first series of experiments a peptide pool matrix was used toidentify epitopes in mice vaccinated with the AdC6-gDPolN vector, butnot challenged with an AAV-1.3HBV vector. A number of regions in thesenaïve mice were identified that elicited potent responses (e.g. greaterthan 1% IFN-γ production CD8+CD44+ T cells. FIG. 7A and FIG. 8A. In thesecond experiment, mice were challenged with 1×10¹⁰ virus genomes (vg)of the AAV-1.3HBV vector, were vaccinated 4 weeks later with an AdC6vector expressing the same HBV polymerase sequence (gDPolN) as in theinitial experiment in non-challenged mice, and ten weeks thereafter theHBV PolN-specific CD8+ T cell epitope profile was determined usingpeptide pool matrices on splenocytes from the mice that had beenchallenged prior to vaccination. FIG. 7B and FIG. 8B. The experiment wasrepeated using more stringent conditions by challenging mice with a1.5×10¹¹ vg dose of the AAV8-1.3HBV vector. Mice were again vaccinated 4weeks later and were tested approximately 10 weeks after vaccination forCD8+ T cell responses to the peptide pool matrices. FIG. 7C and FIG. 8C.In both experiments, compared to the results obtained from unvaccinatedmice, a shift was observed in the epitope profile in AAV8-1.3HBVinfected mice, which at the time of vaccination had high viral loadsbetween 10⁷-10⁹ vg per ml of serum. The effect was more pronounced inmice that had been challenged with a high dose of the AAV8-1.3HBVvector. In both experiments a reduction in responses were observed.Furthermore, especially in mice challenged with the high dose ofAAV8-1.3HBV, the results showed a loss of CD8+ T cells to many of theepitopes that showed immunodominance in uninfected vaccinated mice (e.g.within region represented by peptides 50 to 59, FIG. 8), a betterpreservation of epitopes that were subdominant (such as those within theregion represented by peptides 2 to 8) as well as new epitopes, such asin the region presented by peptides 10 to 29. These data confirm a shiftfrom recognition of dominant to recognition of subdominant epitopes.

Based on these data, a new HBV polymerase N-terminal domain insert (HBVPolN v2) was generated (SEQ ID NO: 173):

HFRKLLLLDEEAGPLEEELPRLADEGLNRRVA EDLNLGNLPEWQTPSFPKIHLQEDIVDRCKQFVGPLIVNEKRRLKLIMPARFYPNVIKYLPLDK GIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRN SKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVTThis insert induced CD8+ T cell responses mainly to subdominant epitopesto which responses remain intact in mice with high HBV viral loads.Immunogenicity and Efficacy of gDCore, gDPolN, and gDPolC Vaccines

The immunogenicity and efficacy the AdC6-gDCore, AdC6-gDPolN,AdC6-gDPolC, AdC7-gDCore, AdC7-gDPolN, and AdC7-gDPolC vaccines in anAAV8-HBV mouse model were analyzed.

Methods—Immunogenicity

C57Bl/6 mice (n=5 per group) were injected with various doses of:AdC6-gDCore (gDCore nucleic acid sequence corresponding to SEQ ID NO:15); AdC6-gDPolN (gDPolN nucleic acid sequence corresponding to SEQ IDNO: 17); or AdC6-gDPolC (gDPolC nucleic acid sequence corresponding toSEQ ID NO: 19). Two months after the first injection, AdC6vector-immunized mice were boosted with AdC7 vectors containing the sameinsert (e.g. AdC7-gDCore, AdC7-gDPolN, or AdC7-gDPolC). Mice were bledat 14 days and 56 days after the injection and T cell frequencies to thevarious HBV inserts were analyzed by intracellular cytokine staining(ICS) for interferon (IFN)-γ upon stimulation of cells with overlappingpeptides representing the HBV sequences. Control cells were culturedwithout peptides. Frequencies and phenotype of CD8+ T cells to oneimmunodominant epitope within PolN were tested for by staining with anMHC I tetramer. The breadth and specificity of CD8+ T cell responses toindividual peptides within a target sequence was performed via epitopemapping of splenocytes (CD8+ T cells tested by ICS for IFN-γ).

To assess CD8+ T cells in the liver, C57Bl/6 mice (n=8 per group)received intravenous administration of 1×10¹⁰ viral genomes (vg) ofAAV8-1.3HBV, 1×10¹¹ vg of AAV8-1.3HBV, or nothing via their tail vein,and 4 weeks later received a single IM injection of 5×10⁹ viralparticles (vp) of AdC6-gDPolN. Eight weeks after the IM injection, micewere sacrificed, livers were removed, and lymphocytes were isolated andstained with T cell markers and a tetramer recognizing the T cellreceptor to an immunodominant epitope present in the PolN sequence.

In a separate experiment, three groups of C57Bl/6 mice (n=4 per group)received a single IM injection of 5×10⁹ vp of AdC6-gDPolN at four weeks(−) or received either intravenous administration of 1×10¹¹ viralgenomes (vg) of AAV8-1.3HBV via their tail vein with or without a singleIM injection of 5×10⁹ vp of AdC6-gDPolN four weeks later. Approximately2 months after administration of AAV8-1.3HBV, mice were sacrificed,livers were removed and liver slices were prepared from each of thethree groups, stained with hematoxylin and eosin and evaluated forlymphocytic infiltrates. From the same experiment, cells were stainedwith a specific tetramer and fluorochrome labeled antibodies to T-bet(clone 4B10, BV785 stain) or antibodies to PD-1 (clone 29F.1A12, BF605stain), TIM-3 (clone RMT3-23, Pe/Cy7 stain), CTLA-4 (clone UC10-4B9, PEstain), or LAG-3 (clone C9B7W, BV650 stain). Cells were analyzed by flowcytometry and gated on CD44+CD8 tetramer positive cells, which were thengated on the markers. Percent marker positive cells were identified fromhistograms in comparison to naïve T cells.

Methods—Efficacy

AAV8-1.3HBV Vector Studies—To assess the impact of AdC6-gDPolN onchronic HBV virus exposure, C57Bl/6 mice (n=8 per group) were challengedintravenously via their tail vein with 1×10¹⁰ vg of AAV8-1.3HBV and fourweeks later immunized with a single IM injection of 5×10⁹ vp ofAdC6-gDPolN. HBV DNA viral titers were evaluated by qPCR; pre- andpost-vaccination changes from baseline (log₁₀ copies/mL) were reported.Viral genome copy numbers were assessed at four, six, eight, ten, andtwelve weeks after AAV8 challenge. Viral dynamics were assessed by PCRover time and the change in log 10 in HBV copies per mL were assessed.The number of mice showing a one, two or three log reductions atdifferent points after treatment was assessed.

Impact of chronic HBV virus exposure on CD8+ T cell antigen recognitionover time—The effect of AAV8-1.3HBV on vaccine-induced hepatic CD8+ Tcells was assessed. The epitope profile in splenocytes of naïve miceimmunized with a single IM injection of 5×10⁹ vp of AdC6-gDPolN wasdetermined 4 weeks after vaccination. Mice challenged with 1×10¹⁰ and1.5×10¹¹ vg of AAV8-1.3HBV and subsequently vaccinated with 5×10⁹ vp ofAdC6-gDPolN 4 weeks later had CD8+ T cell epitope profiles insplenocytes performed 10 weeks after vaccination (14 weeks after AAVinjection). Epitope profiles between AAV-naïve and AAV-treatedvaccinated animals were compared. PolN-specific CD8+ T cells from liverwere analyzed for differentiation markers.

Results

Immunogenicity—Vaccination induced robust and sustained CD8+ T cellresponses to PolN (median frequencies over all circulating CD8+ T cells:6.0%) and lower responses to PolC and core (median frequencies: 1.0% &0.4%, respectively; FIG. 9A and FIG. 9B). Boosting at 8 weeks increasedresponses to all regions with significant changes being observed forcore (p=0.007) (FIG. 9C). FIGS. 9A-9C show % CD8+ T cells over all CD8+T cells for individual mice with medians indicated by the lines.Vaccination induced broad epitope recognition by CD8+ T cells that wasfurther enhanced after boosting (27% to 34%; FIG. 10).

At week 12 following AdC6-gDPolN vaccination, AAV8-1.3HBV-infectedvaccinated mice showed a preferential increase in hepatic CD8+infiltrates (FIG. 11A-11B and FIG. 12A-12F), a decreased presence ofvaccine-induced HBV-specific CD8+ T cells (FIG. 11A and FIG. 11B) andslightly reduced levels of T-bet (suggestive of loss of effectorfunctions) (FIG. 13A-13B). FIG. 11A shows the % CD8+ T cells over allrecovered lymphocytes from individual livers. FIG. 11B shows percenttetramer positive CD8+ cells, which were identified from histograms incomparison to naïve T cells. No clear pattern of cellular markerssuggestive of T cell differentiation to an exhaustion phenotype wasobserved, however, between vaccinated AAV1.3HBV-infected and -uninfectedmice (FIG. 13A-13B).

Efficacy—Following a single IM injection of the AdC6-gDPolN vector,AAV8-1.3HBV-infected mice had multi-log HBV DNA declines in serum thatpersisted throughout the 8-week post vaccination period (FIG. 14). Postvaccination, median declines in serum HBV DNA viral load levels at fourand eight weeks were 0.86 and 2.69 log₁₀ cps/mL, respectively (FIG.14A). At week 8, all animals had a >1 log₁₀ cps/mL, 6/7 (86%) had >2log₁₀ cps/mL, and 2/7 (29%) had >3 log₁₀ cps/mL declines from baseline(FIG. 14B).

Following a single AdC6-gDPolN vector injection, distinct CD8+ T cellrecognition patterns to PolN peptides in splenocytes were observed whenAAV-HBV-infected and naïve mice were compared. FIG. 15A and FIG. 15Billustrate the results from experiments in which mice were firstinjected with AAV-1.3HBV and then four weeks later boosted with 10¹⁰ vpof the AdC6-gDPolN vector, splenocytes were harvested 8 weeks after theimmunization and tested by ICS for IFN-γ upon a short in vitrostimulation with individual peptides spanning the sequence of PolN.Background frequencies obtained without peptide were subtracted. FIG.15A shows the peptide recognition profile of mice that received theAdC6-gDPolN vaccine only followed by those that were first injected withthe indicated doses of the AAV8-1.3HBV vector. The pie graphs in FIG.15B show the corresponding responses to peptides that reached thethreshold of 0.1% of all CD44⁺CD8⁺ cells (data correspond to those inFIG. 15A). Each slice/color represents the frequency of the response toan individual peptide with size showing the proportion of the total;only responses greater than 0.1% were included. Pullouts indicateepitopes only recognized in AAV8-1.3HBV infected mice. It was found thatpre-treatment with AAV reduced both the number of epitopes recognizedafter a single IM prime and the magnitude of the immune response as thesum total of IFN-γ producing CD8+ T cells over the pool of CD8⁺ T cells.AAV pre-treatment shifted the T cell recognition to new epitopes, whichrepresent roughly a third of the detectable CD8⁺ T cell response. Thepercentage of functional HBV-specific CD8+ T cell responses were highestin naïve mice (4.4%, FIG. 15B) but decreased in the presence of low andhigh dose AAV8-1.3HBV (2.0% & 0.6%; respectively, FIG. 15B).AAV8-1.3HBV-uninfected animals showed strong CD8+ T cell responses to anumber of epitopes, which were decreased and shifted in AAV-HBV-infectedanimals to include T cell recognition of new epitopes.

DISCUSSION

An HBV therapeutic vaccine that targets early CD8+ T cell activationusing gD as a genetically encoded checkpoint inhibitor was generated andwas shown to:

-   -   Induce potent and durable CD8+ T cell responses to key HBV        antigens (FIG. 9);    -   Stimulate very broad CD8+ T cell responses (FIG. 10) that        included sub-dominant epitope recognition (FIG. 15); and    -   Achieve sustained multi-log HBV DNA viral load reductions in an        AAV mouse model (FIG. 14) with preferential trafficking of        functional CD8+ T cells to the liver (FIGS. 11 and 12).

In the disclosed AAV studies, AAV-induced HBV infection caused loss ofCD8+ T cell recognition to dominant epitopes of PolN followingvaccination with AdC6-gDPolN (FIG. 15). Without intending to be bound bytheory, it is believed that it is the breadth of the CD8+ T cellsinduced by gD and their ability to recognize subdominant epitopes thatled to a sustained immune response and multi-log suppression of HBV.

Immunogenicity of AdC6/7-gDPolN in Blood and Liver Following Vaccinationin AAV-Induced HBV-Infected Animals

The following studies were performed to evaluate CD8⁺ T cell responsesto the AdC6-gDPolN vaccine in blood, spleens, and livers of animals inthe presence of pre-existing AAV-induced HBV infection.

Experiment #1—CD8⁺ T Cell Responses in AAV8-1.3HBV Infected Mice:Response Kinetics in Blood

Purpose—To assess the effect of sustained titers of HBV antigen on CD8⁺T cell responses to the gDPolN antigen as expressed within the AdC6vector.

Methods—C57Bl/6 mice were injected i.v. with the 10¹⁰ of the AAV8-1.3HBVvector. Four weeks later they were vaccinated with 5×10⁹ vp of theAdC6-gDPolN vector. Control mice received only the AdC6-gDPolN vector.Naïve mice served as additional controls. Mice were boosted 2 monthslater with the same dose of the AdC7-gDPolN vaccine. Blood was collectedat various times after the prime and the boost and PBMCs were tested forIFN-γ-producing CD8⁺ T cells.

Results—As shown in FIG. 16, mice mounted a vigorous PolN-specific CD8⁺T cell response 2 weeks after vaccination, which gradually declined byweek 8 and then increased again after the boost. The CD8⁺ T cellresponse was more stable after the boost than after the prime. At mosttime points tested responses were lower in mice that had been injectedwith the AAV8-1.3HBV vector than in the controls that had not beeninjected with an AAV vector.

Experiment #2—CD8⁺ T Cell Responses in AAV8-1.3HBV Infected Mice:Responses in Liver

Purpose—To assess CD8⁺ T cell responses including markers indicative ofT cell exhaustion in livers of AAV8-1.3HBV-infected, vaccinated mice.

Methods—C57Bl/6 mice were injected i.v. with the 10¹⁰ or 10¹¹ vg of theAAV8-1.3HBV vectors. Four weeks later they were vaccinated with 5×10⁹ vpof the AdC6-gDPolN vector. Control mice received only the AdC6-gDPolNvector. Naïve mice served as additional controls. Mice were boosted 2months later with the same dose of the AdC7-gDPolN vaccine.

To obtain hepatic lymphocytes, livers were cut into small fragments andtreated with 2 mg/ml Collagenase P, 1 mg/ml DNase I (all from Roche,Basel Switzerland) and 2% FBS (Tissue Culture Biologicals, Tulare,Calif.) in L15 under agitation for 1 hour. Liver fragments werehomogenized, filtrated through 70 μm strainers and lymphocytes werepurified by Percoll-gradient centrifugation and washed with DMEMsupplemented with 10% FBS. Lymphocytes were stained with a violetlive/dead dye (Thermo Fisher Scientific), anti-CD8-APC (clone 53-6.7,BioLegend), anti-CD44-Alexa Flour 700 (clone IM7, BioLegend),anti-EOMES-Alexa Fluor 488 (clone Dan1Imag, eBioscience), anti-PD1-BV605(clone 29F.1A12, BioLegend), anti-LAG3-BV650 (clone C9B7W, BioLegend),anti-T-bet-BV786 (clone 4B10, BioLegend), anti-CTLA-4-PE-A (cloneUC10-4B9, BioLegend), anti-TIM-3-Pe-Cy7-A (clone RMT3-23, BioLegend),and an APC-labeled MHC class I tetramer (NIH tetramer Facility, EmoryUniversity, Atlanta Ga.) corresponding to amino acids 396-404 FAVPNLQSL(SEQ ID NO: 188) (peptide 55) of the HBV polymerase at +4° C. for 30 minin the dark. Cells were washed and were analyzed by a BD FACS Celesta(BD Biosciences, San Jose, Calif.) and DiVa software. Post-acquisitionanalyses were performed with FlowJo (TreeStar, Ashland, Oreg.).

Results—The frequencies of CD8⁺ T cells within the lymphocytic liverinfiltrates were analyzed. Frequencies of CD8⁺ T cells within thelymphocytic liver infiltrates were increased in vaccinated mice ascompared to naïve mice, and further increases were seen in mice thatprior to vaccination had been injected with the AAV8-1.3HBV vector (FIG.17A). Frequencies of PolN-specific CD8⁺ T cells identified by stainingwith a tetramer specific for an epitope present in the PolN insert werereduced in AAV-1.3HBV-injected mice (FIG. 17B).

The phenotypes of the infiltrating tetramer⁺CD8⁺ T cells in comparisonto naïve (i.e., tetramer⁻CD44⁻CD8⁺ T cells) were assessed by determiningthe mean fluorescent intensity of a dye linked to a given antibody (FIG.18A-FIG. 18F) and by assessing the percentages (FIG. 19A-FIG. 19F) ofCD8⁺ T cells that were positive for the indicated markers.

T-bet which controls a number of CD8⁺ T cell functions, was reduced onhepatic CD8⁺ T cells from mice that had been injected with AAV8-1.3HBVprior to vaccination in comparison the vaccine only group. Exhaustionmarkers were not increased in AAV8-1.3HBV-pre-treated groups suggestingthat the observed loss of PolN-specific CD8⁺ T cells in presence of HBVwas unlikely to be caused by classical CD8⁺ T cell exhaustion (FIG.18A-FIG. 18F and FIG. 19A-FIG. 19F).

Experiment #3—Breadth of the PolN-Specific CD8+ T Cell Response inAAV8-1.3HBV Infected Mice

Purpose—To assess if the presence of HBV affects the breadth of the CD8⁺T cell response to PolN expressed within gD by the AdC vaccines.

Methods—Mice were injected i.v. with the 10¹⁰ or 10¹¹ vg of theAAV8-1.3HBV vectors and were boosted 2 months later with thecorresponding AdC7 vectors. Control mice received only the AdC6-gDPolNvector. Mice were euthanized 10 weeks later and the pooled splenocyteswere tested against pools of peptides in the non-AAV infected animalstudy. Results are provided in FIG. 20A-FIG. 20C.

In a second experiment, mice were injected i.v. with the 10¹⁰ or 10¹¹ vgof the AAV8-1.3HBV vectors. Four weeks later they were vaccinated with5×10¹⁰ vp of the AdC6-gDPolN vector. Control mice received only theAdC6-gDPolN vector. Naïve mice served as additional controls.Splenocytes were analyzed 6 weeks later for IFN-γ-producing CD8⁺ T cellsin response to individual peptides spanning the PolN sequence. Resultsare provided in FIG. 20D-FIG. 20F.

Results—The presence of HBV, especially high titers of HBV such as afterinjection with the 10¹¹ vg dose of AAV8-HBV1.3, not only reduced overallCD8⁺ T cell responses to the PolN sequence as presented by theAdC6-gDPolN vaccine but also caused a shift in the epitope recognitionprofile.

Experiment #4—Functions of Hepatic PolN-Specific CD8+ T Cells inAAV8-1.3HBV Infected Mice

Purpose—To evaluate if liver-infiltrating PolN-specific CD8⁺ T cellsremain functional in AAV8-1.3HBV infected mice.

Methods—In the first experiment, C57BL/6 mice were injected i.v. with3×10¹¹ vg of AAV8-1.3HBV. One group was vaccinated 8 weeks later with5×10¹⁰ vp of AdC6-gDPolN vector. The other group was left unvaccinated.Mice were euthanized 4.5 months later and splenocytes were tested forfrequencies of CD8⁺ T cells producing IFN-γ in response to the PolNpeptide pool.

In the second experiment, mice were injected with graded concentrationsof AAV8-1.3HBV (1×10¹⁰, 4×10¹⁰, or 1×10¹¹). All mice were vaccinated 4weeks later with 5×10¹⁰ vp of the AdC6-gDPolN vector. The mice wereboosted 2 months later with the same dose of the AdC7-gDPolN vector.Mice were euthanized 2 months later and lymphocytes were isolated fromlivers and tested for CD8⁺ T cells producing IFN-γ in response to thePolN peptide pool. Cells were also stained with an antibody to Tox, atranscription factor that increases in exhausted T cells.

Results—As shown in FIG. 21, vaccine-induced CD8⁺ T cells remainedfunctional in mice that had been injected with the AAV8-1.3HBV vector.

Experiment #5—Effect of Vaccination of AAV8-1.3HBV Infected Mice onLiver Histology

Purpose—To assess if AdC6/7-gDPolN vaccination ofAAV.8-1.3HBV-vaccinated mice causes sustained liver damage.

Methods—Mice were injected with 10¹⁰ vg of the AAV8-1.3HPV given i.v.One month later they were vaccinated with 5×10⁹ vp of the AdC6-gDPolNvector. The mice were boosted 2 months later with the same dose of theAdC7-gDPolN vector given at the same dose. The mice were euthanized ˜2months later. Liver sections were collected and fixed in 10%formaldehyde. Sections (˜3 μm in thickness) were prepared and stainedwith Hematoxylin Eosin (H&E). They were reviewed under a lightmicroscope at 20× magnification.

Results—One out of 33 sections from mice that had received both the AAVvector and the vaccine showed a small lymphocytic infiltrate that was atthe margin of the liver section.

As shown in FIG. 21B, following a single gDPolN vaccination in HLA-A2-tgmice, frequencies of IFN-γ producing hepatic CD8⁺ T cells were reducedin mice receiving AAV as compared to those that had only beenvaccinated.

Conclusions

-   -   CD8⁺ T cell responses to PolN were reduced in AAV8-1.3HBV        infected mice. Nevertheless, they remained detectable.    -   Exhaustion markers were not increased in AAV8-1.3HBV pre-treated        animals suggesting that the observed loss of PolN-specific CD8⁺        T cells in the presence of HBV was unlikely to be caused by        classical CD8⁺ T cell exhaustion.    -   AAV-induced HBV-infection caused a shift in the epitope        recognition profile of CD8⁺ T cell responses to PolN.    -   Vaccine-induced CD8⁺ T cells remained functional in mice that        had been previously infected with the AAV8-1.3HBV vector.    -   The vaccine used in a prime boost regimen did not cause overt        liver damage in HBV positive mice.

Generation of HBV PolN-PolC-Core Constructs

Two multi-antigen inserts (second generation PolN-PolC-Core and thirdgeneration PolN-PolC-Core) were generated. The sequences of theseinserts are shown below:

2nd generation HBV vaccine insert (“HBV2”)(Pol N (italics)-Pol C (underlined)-Core) (SEQ ID NO: 174)YIPLDKGIKPYYPEHAVNHYFQTRHYLHTLWK AGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCITHLVNILEDWGPCDEHG EHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGITRVSWPKFAVPNIQSLTNLLS SNLSWESLDV QAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQRMRGT FVAPLPIHTAELLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPS ALNPADDVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIRT PPAYRPPNAPILSTLPETTVVRRRDRGR3rd generation HBV vaccine insert (“HBV3”)(Pol N (italics)-Pol C (underlined)-Core) (SEQ ID NO: 175)HFRKLLLLDEEAGPLEEELPRLADEGLNRRVA EDLNIGNLPEWQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDK GIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRN SKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVT QAFTFSPTYKAFLSKQYLNLYPVA RQRPGLCQVFADATPTGWGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTDNSVV LSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDVGSNLEDPASRELVVSYVNVNMGLKIR QLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGR

The second generation HBV (“HBV2”) insert includes immuno-dominant PolNepitopes identified from mice that had not been infected with theAAV8-1.3HBV vector prior to vaccination. Many of these epitopes werefound to be lost in a mouse model of chronic HBV infection brought aboutby pre-administering an AAV8-1.3HBV vector (as defined in “EpitopeShifting” above). The third generation HBV (“HBV3”) insert selects forcontiguous regions of PolN that were preferentially recognized by micewith high loads of HBV (see above). Regions of Core and PolC wereselected for both constructs using the following general formula:regions with the highest immune responses on either prime (FIG. 3) orboost (FIG. 5) regions in C57Bl/6, BALBc and HLA-A2 tg mice, and withthe aim of selecting a large contiguous region instead of selectingunique epitopes and inserting spacer sequences between them.

Genetic Integrity and Stability of the 2^(nd) and 3^(rd) Generation HBVInserts (HBV2 and HBV3)

Western Blot—Purified recombinant viral vector preparations(AdC6-gDHBV2, AdC6-gDHBV3, AdC7-gDHBV2, and AdC7-gDHBV3) were evaluatedfor their ability to elicit transgene-product expression in vitro. Tothat end, Western Blot assays were performed to assess the expression ofgD protein in cell lysates following cell culture infection with thevector of interest. Adherent HEK293 cell monolayers were infected withknown quantities of the purified vector and harvested at 48 hourspost-infection, resuspended in lysis and extraction buffer containingprotease inhibitors, and lysed by sonication. The total protein extractswere denatured by the use of dithiothreitol as a redox agent andsubmitted to electrophoresis in a 12% Bis-Tris polyacrylamide gel(PAGE). Subsequent to protein separation by SDS-PAGE, the samples weretransferred onto an activated polyvinylidene difluoride membrane by wetelectrophoretic transfer. The membrane was immunostained for thedetection of gD protein using the primary antibody to gD diluted to1:1000 in saline (clone PA1-30233, Invitrogen, Carlsbad, Calif.) for 1 hat room temperature. Membranes were washed with 1×TBS-T prior toincubating with HRP-conjugated goat anti-rabbit secondary IgG (ab6721,Abcam, Cambridge UK) for 1 h at room temperature. This was followed bythe addition of a luminol-based chemiluminescent substrate. The stainedmembrane was exposed to an autoradiography film and signal emission wasevaluated after processing by an automated film developer. Followingdocumentation of the gD protein expression in infected HEK293 celllysates, the membrane was stripped and re-probed for the presence ofR-actin in the total protein extract samples. This staining step wasemployed to evaluate the consistency of the PAGE sample loading step andthus better support the semi-quantitative analysis of the in vitrostimulation of gD protein expression by the recombinant viral vector.

Stability—To ensure the genetic integrity of the viral construct, thegenetic stability of each recombinant viral vector lot was assessedthrough sequential viral passages in adherent HEK293 cell cultures. Therecombinant virus pool resulting from each transfection was culturedunder standard growing conditions for a total of 12 passages. In thelast passage, the virus pool was expanded and the crude harvest purifiedby cesium chloride gradient. Following vector purification, viral DNAwas isolated using the QIAGEN DNeasy Blood & Tissue Kit and evaluated byrestriction enzyme digest with Ase I and Bgl II, two restriction enzymesthat cleave the DNA template in distinct construct-specific pre-definedbanding patterns. After digestion, samples were submitted toelectrophoresis in 1% agarose gel containing ethidium bromide to allowfor the visualization of the digested bands, followed by documentationof results using a digital gel imaging system. Viral preparations thatexhibited banding patterns identical to those of an early passage viruswere considered to have maintained the original molecular clonestructure and thus deemed stable at the end of 12 viral passages.

Results—The banding patterns of viral vector DNAs remained stable after12 passages compared to that after 5 passages indicating the vectorgenomes were stable (data not shown).

Immunogenicity of the 2^(rd) and 3^(rd) generation HBV inserts (HBV2 andHBV3) as expressed by AdC6 or AdC7 Vectors

Purpose—To assess CD8⁺ T cell responses to the HBV2 and HBV3 insertsexpressed by AdC6 vectors or AdC7 vectors.

Methods—Groups of C57Bl/6 mice were injected with 5×10⁹ or 5×10¹⁰ vp ofAdC6-gDHBV2 or AdC6-gDHBV3 vector. Mice injected with the same doses ofthe AdC6-gDPolN vector served as positive controls; naïve mice served asnegative controls. Mice were bled 14 days later and PBMCs were testedfor frequencies of CD8⁺ T cells producing IFN-γ in response to peptidepools corresponding to the HBV inserts. Four weeks later (6 weeks aftervaccination) mice were bled again and tested with the PolN-specifictetramer. AdC6-gDHBV3 immunized mice were excluded as this insert lacksthe epitope that corresponds to the tetramer.

Groups of C57Bl/6 mice were injected with 5×10⁹ or 5×10¹⁰ vp ofAdC7-gDHBV2 or 5×10¹⁰ vp of AdC7-gDHBV3 vector. Naïve mice served asnegative controls. Mice were bled 14 days later and PBMCs were testedfor frequencies of CD8⁺ T cells producing IFN-γ in response to peptidepools corresponding to the HBV inserts.

Immunogenicity of AdC7 Prime AdC6 Boost

Mice were bled ˜4 weeks later and PBMCs were retested by ICS for CD8⁺ Tcells producing IFN-γ and/or TNF-α in response to the peptides for theinserts. Mice were boosted two months after the prime with the same doseof the heterologous vector expressing the same insert. PBMCs were testedby ICS 2 weeks later and pre- and post-boost CD8⁺ and CD4⁺ T cellresponses were compared. The AdC7-gDHBV2 vector induced robustfrequencies of CD8⁺ T cells producing IFN-γ and/or TNF-α after theprime. Frequencies increased after the AdC6-gDHBV2 boost and this wasespecially pronounced after the low vector doses and for CD8⁺ T cellsproducing IFN-γ. The AdC7-gDHBV3 vector was poorly immunogenic but CD8⁺T cell responses became positive after the AdC6-gDHBV3 boost. In thesame token CD4+ T cell responses were marginal after the prime butincreased after the boost. There was no marked difference in CD4responses to the HBV2 or HBV3 insert.

Conclusions

-   -   Both the AdC6-gDHBV2 and AdC7-gDHBV2 vectors were highly        immunogenic (FIG. 22A, FIG. 22B, and FIG. 23) and responses        increased after a boost with a heterologous AdC vector        expressing the same insert (FIG. 24).    -   The AdC7-gDHBV2 and AdC7-gDHBV3 vectors displayed borderline        immunogenicity consistent with their design as they lack the        epitope that corresponds to the tetramer being used (FIG. 22A        and FIG. 23).    -   Boosting AdC7-gDHBV2 with AdC6-gDHBV2 enhances CD8⁺ T cell        responses.

Comparison of HBV DNA Viral Titers in AdC6-gDPolN, AdC6-gDHBV2,AdC6-gDHBV3, or AdC6-HBV2 AAV-Infected Mice Methods

Five groups of C57Bl/6 mice were challenged with 1×10⁹ vg of AAV8-1.3HBVand were vaccinated 4 weeks later with 1×10¹⁰ vp of either AdC6-gDPolN(n=10), AdC6-gDHBV2 (n=10), AdC6-gDHBV3 (n=10), or AdC6-HBV2 without gD(n=10); AAV-infected, non-vaccinated animals (“naive”) (n=10) andnon-AAV-infected, non-vaccinated animals (n=2-5) served as controls.Viral titers were tested 4 weeks after AAV injection (beforevaccination) and compared to levels 4 weeks after vaccination (week 8after AAV injection).

Results

At week 8, the median HBV viral titers increased by 0.98 log₁₀ cps/mL innaïve mice, remained unchanged in AdC6-HBV2 vaccinated mice, anddeclined by −0.04, −1.09 and −2.13 log₁₀ cps/mL in AdC6-gDHBV3,AdC6-gDPolN and AdC6-gDHBV2 vaccinated animals, respectively (FIG. 25A).The results for individual mice are shown in FIG. 25B—all AdC6-gDPolNand AdC6-gDHBV2 vaccinated animals had greater than 1 and 2 log₁₀copies/mL declines, respectively; in contrast, none of the naïve,AdC6-HBV2, or AdC6-gDHBV3 vaccinated animals had a 1 log₁₀ copies/mL orgreater decline at Week 8.

Immunogenicity Studies for gDHBV2 and gDHBV3

The induction of CD8⁺ T cell responses and their breadth to segments ofHBV core and polymerase contained in either gDHBV2 or gDHBV3 following asingle prime injection or prime followed by a boost vaccination with aheterologous vector containing the same insert were evaluated.

Experiment 1

Purpose: Assess IFN-γ⁺CD8⁺ T cell responses following prime and boostvaccinations with gD-HBV2 and gD-HBV3 expressed by heterologouschimpanzee adenoviral vectors (AdC6 and AdC7) in C57Bl/6 mice.

Methods: Four groups of five C57Bl/6 mice were immunized viaintramuscular injection as follows: (a) 5×10¹⁰ vp AdC7-gDHBV2 followedtwo months later by 5×10¹⁰ vp AdC6-gDHBV2; (b) 5×10⁹ vp AdC7-gDHBV2followed two months later by 5×10⁹ vp AdC6-gDHBV2; (c) 5×10¹⁰ vpAdC7-gDHBV3 followed two months later by 5×10¹⁰ vp AdC6-gDHBV3; or (d)no vaccine. Blood was assessed by ICS for IFN-γ⁺CD8⁺ T cell responses 2and 6 weeks after the prime, prior to the boost, and then 2 and 4 weeksafter the boost.

Results: At all time points tested each vaccine construct was found toinduce IFN-γ⁺ CD8⁺ T cells. FIG. 26 shows the percent of parental IFN-γand/or TNF-α producing CD8⁺ T cells (FIG. 26A), CD44+CD8+ T cells (FIG.26B), CD4+ T cells (FIG. 26C) or CD44+CD4+ T cells (FIG. 26D). Immuneresponses as assessed by ICS from PBMCs of individual mice are shown twoand eight weeks after the prime, as well as two and four weeks after theboost as the mean.

Experiment 2

Purpose: Compare IFN-γ⁺CD8⁺ T cell responses following different dosesof prime and boost vaccinations with gD-HBV2 and gD-HBV3 to that withgD-PolN using heterologous chimpanzee adenoviral vectors (AdC6 and AdC7)in C57Bl/6 mice.

Methods: Groups of C57Bl/6 mice (n=5 mice/group) were immunized asfollows:

gDPolN Groups

-   -   (a) 5×10⁹ vp AdC6-gDPolN followed three months later by 5×10⁹ vp        AdC7-gDPolN; and    -   (b) 5×10¹⁰ vp AdC6-gDPolN followed three months later by 5×10¹⁰        vp AdC7-gDPolN

gDHBV2 Groups

-   -   (c) 5×10⁹ vp AdC6-gDHBV2 followed three months later by 5×10⁹ vp        AdC7-gDHBV2 and;    -   (d) 5×10¹⁰ vp AdC6-gDHBV2 followed three months later by 5×10¹⁰        vp AdC7-gDHBV2

gDHBV3 Groups

-   -   (e) 5×10⁹ vp AdC6-gDHBV3 followed three months later by 5×10⁹ vp        AdC7-gDHBV3 and;    -   (f) 5×10¹⁰ vp AdC6-gDHBV3 followed three months later by 5×10¹⁰        vp AdC7-gDHBV3

No Treatment Served as Controls

For all treatment groups, immunogenicity CD8⁺ T cell responses was fromblood assessed by ICS for IFN-γ⁺ at two and six weeks after the prime,prior to the boost, and then two and six weeks after the boost.Immunogenicity was also assessed by tetramer staining using anAPC-labeled MHC class I tetramer (NIH tetramer Facility, EmoryUniversity, Atlanta Ga.) corresponding to amino acids 396-404 FAVPNLQSL(peptide 55) of the HBV polymerase at week four after the prime. HBV3does not contain the FAVPNLQSL peptide.

Results: At all time points, each vaccine tested was found to induceIFN-γ+CD8⁺ T cells. Results obtained with the gDHBV2 vaccine weresimilar to those obtained with the gDPolN vaccine; the gDHBV3 vaccinewas less immunogenic. Upon tetramer staining, frequencies of thespecific CD8⁺ T cells were comparable between the two vaccines; a numberof activation markers tended to be more highly expressed ontetramer+CD8+ T cells from the gDHBV2-immunized groups. FIG. 27 showsCD8⁺ T cells at multiple time points: four weeks after prime (FIG. 27A);two weeks after the boost (FIG. 27B); and four weeks after the boost(FIG. 27C). The graph shows the overall frequencies of CD8⁺ T cellsproducing IFN-γ⁺ as assessed by ICS.

FIG. 28 shows cytokine-producing CD4+ T cells at multiple time points:four weeks after prime (FIG. 28A); two weeks after the boost (FIG. 28B);and four weeks after the boost (FIG. 28C) as assessed by ICS. The dashedline indicates the cut-off for positive responses, based on the resultsfrom the naïve mice.

FIG. 29 shows the results of tetramer staining gated on either CD8+ Tcells (FIG. 29A) or CD44+CD8+ T cells (FIG. 29B) at four weeks after theprime.

FIG. 30 shows the phenotypes of the tetramer+CD8+ T cells shown as themean fluorescent intensity of a dye linked to the indicated antibody:FIG. 30A anti-PD1 antibody conjugated to BV605; FIG. 30B anti-LAG3antibody conjugated to BV650; FIG. 30C anti-TIM3 antibody conjugated toPe-Cy7-A; FIG. 30D anti-CTLA4 antibody conjugated to PE-A; FIG. 30Eanti-EOMES antibody conjugated to AF488; and FIG. 30F anti-T-betantibody conjugated to BV786.

Experiment 3

The breadth of responses were assessed from pooled splenocytes ofvaccinated C57BL/6 mice which were tested by ICS against the individualpeptides present in the HBV vaccine inserts.

Methods: Four groups of five C57Bl/6 mice were immunized viaintramuscular injection as follows: (a) 5×10¹⁰ vp AdC7-gDHBV2 followedtwo months later by 5×10¹⁰ vp AdC6-gDHBV2; (b) 5×10⁹ vp AdC7-gDHBV2followed two months later by 5×10⁹ vp AdC6-gDHBV2; (c) 5×10¹⁰ vpAdC7-gDHBV3 followed two months later by 5×10¹⁰ vp AdC6-gDHBV3; or (3)no vaccine. Animals were sacrificed eight weeks after the boost andpooled splenocytes were assessed by ICS for IFN-γ⁺ CD8⁺ T cell responsesto individual HBV2 or HBV3 peptides (cut-off for positive responses setat 0.1%).

Results: Independent of the dose, the prime boost regimen with thegDHBV2 vaccines induced responses to several epitopes within core andpolymerase. FIG. 31 shows the CD8⁺ T cell responses after a primevaccination of 5×10¹⁰ vp AdC7-gDHBV2 followed two months later byvaccination with 5×10¹⁰ vp AdC6-gDHBV2. Numbers on the X axis correspondto the SEQ ID NO as provided herein. FIG. 32 shows the CD8+ T cellresponses after a prime vaccination with 5×10⁹ vp AdC7-gDHBV2 followedtwo months later by vaccination with 5×10⁹ vp AdC6-gDHBV2. Numbers onthe X axis correspond to the SEQ ID NO as provided herein. FIG. 33 showsthe immunogenicity after a prime vaccination with 5×10¹⁰ vp AdC7-gDHBV3followed two months later by vaccination with 5×10¹⁰ vp AdC6-gDHBV3.Numbers on the X axis correspond to the SEQ ID NO as provided herein.

Experiment 4

The breadth of responses were assessed from pooled splenocytes ofvaccinated BALB/c mice which were tested by ICS against the individualpeptides present in the HBV vaccine inserts.

Methods: Five groups of five BALB/c mice were immunized viaintramuscular injection as follows: (a) 5×10¹⁰ vp AdC6-gDHBV2; (b)5×10¹⁰ vp AdC6-gDHBV3; (c) 5×10¹⁰ vp AdC7-gDHBV2; (d) 5×10¹⁰ vpAdC7-gDHBV3; or (e) no vaccine. 12 weeks post vaccination animals weresacrificed, spleens were collected and pooled splenocytes were assessedby ICS for IFN-γ⁺CD8⁺ T cell responses to individual HBV2 or HBV3peptides (cut-off for positive responses set at 0.1%).

Results: At week 12, each vaccine construct was found to be immunogenicacross multiple regions of the Core and Polymerase genes delivered bythe vaccine. FIG. 34 shows the immunogenicity of the AdC6-gDHBV2 andAdC7-gDHBV2 vaccines corresponding to the SEQ ID NO (X axis) as providedherein. Core, PolC, and PolN regions in both HBV2 constructs wereimmunogenic. FIG. 35 shows the immunogenicity of the AdC6-gDHBV3 andAdC7-gDHBV3 vaccines corresponding to the SEQ ID NO (X axis) as providedherein. Core, PolC, and PolN regions in both HBV3 constructs wereimmunogenic.

Experiment 5

Methods: Five groups of C57Bl/6 mice were challenged with 1×10⁹ vg ofAAV8-1.3HBV and were vaccinated 4 weeks later (“prime vaccination”) with1×10¹⁰ vp of either AdC6-gDPolN (n=10), AdC6-gDHBV2 (n=10), AdC6-gDHBV3(n=10), or AdC6-HBV2 without gD (n=10); AAV-infected, non-vaccinatedanimals (n=10) and non-AAV-infected, non-vaccinated animals (n=2-5)serve as controls. Mice will be bled at various times after theinjection and frequencies of insert-specific CD8+ and CD4+ T cells willbe determined by intracellular cytokine staining (ICS) for IFN-γ. PCRwill be performed at 2 weeks, 6 weeks, and 8 weeks after the primevaccination, and a T cell assay will be performed at 4 weeks after theprime vaccination.

At 8 weeks following the prime vaccination, mice will be boosted withAdC7 vectors containing the same antigenic insert used in the primevaccination (“boost vaccination”) and blood and serum will be tested forCD8+/CD4+ T cell as previously described at different time points aftervaccination. PCR will be performed at 2 weeks, 6 weeks, and 10 weeksafter the boost vaccination, and a T cell assay will be performed at 4weeks and 12 weeks after the boost vaccination.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is, therefore, intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

The disclosures of each patent, patent application, and publicationcited or described in this document are hereby incorporated herein byreference, in their entirety.

TABLE 9 Sequences Sequence Genotype AMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASAL ConsensusYREALESPEHCSPHHTALRQAILCWGELMTLATWVGN (SEQ ID NO: 1)NLeDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC Genotype BMDIDpYKEFGASvELLSFLPSDFFPSiRDLLDTAsAL ConsensusYREALESPEHCSPHHTALRQAIlCWGELMNLATWVGS (SEQ ID NO: 2)NLeDPASRELVVsYVNVNMGLKiRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPpAYRPpNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSREsQC Genotype CMDIDpYKEFGASVELLSFLPSDFFPSIRDLLDTASAL ConsensusYREALESPEHCSPHHTALRQAILCWGELMNLATWVGS (SEQ ID NO: 3)NLEDPASRELVVsYVNVNMGLKiRQlLWFHISCLTFGRETVLEYLVSFGVWIRTPpAYRPPNAPILSTLPETTV VRRRGRSPRRRTPSPRRRRSQSPRRRSQSRESQCGenotype D MDIDPYKEFGAtVELLSFLPsDFFPSVRDLLDTASALYReAL ConsensusESPEHCSPHHTALRQAILCWGeLMtLATWVGgNLEDPaSRDL (SEQ ID NO: 4)VVSYVNTNmGLKFRQLLWFHISCLTFGReTViEYLVSFGVWIRTPpAYRPPNAPILSTLPETTVvRRRGRSPRRRTPSPRRRRS QSPRRRRSQSRESQC Initial CoreMDID P YKEFGAX ₁VELLSFLPSDFFPSX ₂DLLDTASALYREA sequenceLESPEHCSPHHTALRQAILCWGELMX ₃LATWVGX ₄NLeDPAS (SEQ ID NO: 5) RX ₅LVVX₆YVNX ₇NMGLKX ₈RQLLWFHISCLTFGRETVX ₉EY LVSFGVWIRTP P AYRP PNAPILSTLPETTVVRRRX ₁₀ X ₁₁GR SPRRRTPSPRRRRSQSPRRRRSQSRESQC Epitope-DIDPYKEFGATVELLSFLPSDFFPSIRDLLDTASALYREALE optimized CoreSPEHCSPHHTALRQAILCWGELMTLATWVGSNLEDPASRELV amino acidVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIR sequenceTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRR (SEQ ID NO: 6)SQSPRRRRSQSRESQC Epitope- GACATCGACCCCTACAAGGAGTTCGGCGCCACCGTGGAGCTGoptimized Core CTGAGCTTCCTGCCCAGCGACTTCTTCCCCAGCATCAGGGAC nucleotideCTGCTGGACACCGCCAGCGCCCTGTACAGGGAGGCCCTGGAG sequenceAGCCCCGAGCACTGCAGCCCCCACCACACCGCCCTGAGGCAG (SEQ ID NO: 7)GCCATCCTGTGCTGGGGCGAGCTGATGACCCTGGCCACCTGGGTGGGCAGCAACCTGGAGGACCCCGCCAGCAGGGAGCTGGTGGTGAGCTACGTGAACGTGAACATGGGCCTGAAGATCAGGCAGCTGCTGTGGTTCCACATCAGCTGCCTGACCTTCGGCAGGGAGACCGTGATCGAGTACCTGGTGAGCTTCGGCGTGTGGATCAGGACCCCCCCCGCCTACAGGCCCCCCAACGCCCCCATCCTGAGCACCCTGCCCGAGACCACCGTGGTGAGGAGGAGGGACAGGGGCAGGAGCCCCAGGAGGAGGACCCCCAGCCCCAGGAGGAGGAGGAGCCAGAGCCCCAGGAGGAGGAGGAGCCAGAGCAGGGAGAGC CAGTGC Epitope-PLSYQHFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNL optimizedGNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFPKIHLQ polymerase N-EDIVDRCKQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDK terminal aminoGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFC acid sequenceGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWG (SEQ ID NO: 8)PCDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGITRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAF YHIPLHPAAMP Epitope-CCCCTGAGCTACCAGCACTTCAGGAAGCTGCTGCTGCTGGAC optimizedGAGGAGGCCGGCCCCCTGGAGGAGGAGCTGCCCAGGCTGGCC polymerase N-GACGAGGGCCTGAACAGGAGGGTGGCCGAGGACCTGAACCTG terminusGGCAACCTGAACGTGAGCATCCCCTGGACCCACAAGGTGGGC nucleotideAACTTCACCGGCCTGTACAGCAGCACCGTGCCCGTGTTCAAC sequenceCCCGAGTGGCAGACCCCCAGCTTCCCCAAGATCCACCTGCAG (SEQ ID NO: 9)GAGGACATCGTGGACAGGTGCAAGCAGTTCGTGGGCCCCCTGACCGTGAACGAGAAGAGGAGGCTGAAGCTGATCATGCCCGCCAGGTTCTACCCCAACGTGACCAAGTACCTGCCCCTGGACAAGGGCATCAAGCCCTACTACCCCGAGCACGCCGTGAACCACTACTTCCAGACCAGGCACTACCTGCACACCCTGTGGAAGGCCGGCATCCTGTACAAGAGGGAGACCACCAGGAGCGCCAGCTTCTGCGGCAGCCCCTACAGCTGGGAGCAGGAGCTGCAGCACGGCAGCTGCTGGTGGCTGCAGTTCAGGAACAGCAAGCCCTGCAGCGAGTACTGCCTGACCCACCTGGTGAACCTGCTGGAGGACTGGGGCCCCTGCGACGAGCACGGCGAGCACCACATCAGGATCCCCAGGACCCCCGCCAGGGTGACCGGCGGCGTGTTCCTGGTGGACAAGAACCCCCACAACACCGCCGAGAGCAGGCTGGTGGTGGACTTCAGCCAGTTCAGCAGGGGCATCACCAGGGTGAGCTGGCCCAAGTTCGCCGTGCCCAACCTGCAGAGCCTGACCAACCTGCTGAGCAGCAACCTGAGCTGGCTGAGCCTGGACGTGAGCGCCGCCTTCTACCACATCCCCCTG CACCCCGCCGCCATGCCC Epitope-HLLVGSSGLSRYVARLSSNSRIINHQHGTMQNLHDSCSRNLY optimizedVSLLLLYKTFGRKLHLYSHPIILKTKRWGYSLNFMGYVIGSW polymerase C-GSLPQDHIIQKIKECFRKLPVNRPIDWKVCQRIVGLLGFAAP terminal aminoFTQCGYPALMPLYACIQSKQAFTFSPTYKAFLSKQYLNLYPV acid sequenceARQRPGLCQVFADATPTGWGLAMGHQRMRGTFVAPLPIHTAE (SEQ ID NO: 10)LLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDPSRGRLGLSRPLLRLPFRPTTGRT SLYAVSPSV Epitope-CACCTGCTGGTGGGCAGCAGCGGCCTGAGCAGGTACGTGGCC optimizedAGGCTGAGCAGCAACAGCAGGATCATCAACCACCAGCACGGC polymerase C-ACCATGCAGAACCTGCACGACAGCTGCAGCAGGAACCTGTAC terminalGTGAGCCTGCTGCTGCTGTACAAGACCTTCGGCAGGAAGCTG nucleotideCACCTGTACAGCCACCCCATCATCCTGAAGACCAAGAGGTGG sequenceGGCTACAGCCTGAACTTCATGGGCTACGTGATCGGCAGCTGG (SEQ ID NO: 11)GGCAGCCTGCCCCAGGACCACATCATCCAGAAGATCAAGGAGTGCTTCAGGAAGCTGCCCGTGAACAGGCCCATCGACTGGAAGGTGTGCCAGAGGATCGTGGGCCTGCTGGGCTTCGCCGCCCCCTTCACCCAGTGCGGCTACCCCGCCCTGATGCCCCTGTACGCCTGCATCCAGAGCAAGCAGGCCTTCACCTTCAGCCCCACCTACAAGGCCTTCCTGAGCAAGCAGTACCTGAACCTGTACCCCGTGGCCAGGCAGAGGCCCGGCCTGTGCCAGGTGTTCGCCGACGCCACCCCCACCGGCTGGGGCCTGGCCATGGGCCACCAGAGGATGAGGGGCACCTTCGTGGCCCCCCTGCCCATCCACACCGCCGAGCTGCTGGCCGCCTGCTTCGCCAGGAGCAGGAGCGGCGCCAAGATCCTGGGCACCGACAACAGCGTGGTGCTGAGCAGGAAGTACACCAGCTTCCCCTGGCTGCTGGGCTGCGCCGCCAACTGGATCCTGAGGGGCACCAGCTTCGTGTACGTGCCCAGCGCCCTGAACCCCGCCGACGACCCCAGCAGGGGCAGGCTGGGCCTGAGCAGGCCCCTGCTGAGGCTGCCCTTCAGGCCCACCACCGGCAGGACC AGCCTGTACGCCGTGAGCCCCAGCGTGN-terminal HSV MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRF gD sequenceRGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYY (SEQ ID NO: 12)AVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFR Signal peptideMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSA in italicsVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPEN QRTVAVYSLKIAGWHGPC-terminal HSV GPKAPYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVG gD sequenceTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSL (SEQ ID NO: 13)LAALVICGIVYWMHRRTRKAPKRIRLPHIREDDQPSSHQPLF Y gDCore aminoMGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRF acid sequenceRGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYY Core underlinedAVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFR (SEQ ID NO: 14)MGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSA Signal peptideVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRA in italicsKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPDIDPYKEFGATVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQCGPKAPYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMHRRTRKAPKRIRLPHIREDDQPSSHQPLFY gDCore nucleicatggggggggctgccgccaggttgggggccgtgattttgttt acid sequencegtcgtcatagtgggcctccatggggtccgcggcaaatatgcc Core underlinedttggcggatgcctctctcaagatggccgaccccaatcgcttt (SEQ ID NO: 15)cgcggcaaagaccttccggtcctggaccagctgaccgaccctccgggggtccggcgcgtgtaccacatccaggcgggcctaccggacccgttccagccccccagcctcccgatcacggtttactacgccgtgttggagcgcgcctgccgcagcgtgctcctaaacgcaccgtcggaggccccccagattgtccgcggggcctccgaagacgtccggaaacaaccctacaacctgaccatcgcttggtttcggatgggaggcaactgtgctatccccatcacggtcatggagtacaccgaatgctcctacaacaagtctctgggggcctgtcccatccgaacgcagccccgctggaactactatgacagcttcagcgccgtcagcgaggataacctggggttcctgatgcacgcccccgcgtttgagaccgccggcacgtacctgcggctcgtgaagataaacgactggacggagattacacagtttatcctggagcaccgagccaagggctcctgtaagtacgccctcccgctgcgcatccccccgtcagcctgcctctccccccaggcctaccagcagggggtgacggtggacagcatcgggatgctgccccgcttcatccccgagaaccagcgcaccgtcgccgtatacagcttgaagatcgccgggtggcacgggcccgacatcgacccctacaaggagttcggcgccaccgtggagctgctgagcttcctgcccagcgacttcttccccagcatcagggacctgctggacaccgccagcgccctgtacagggaggccctggagagccccgagcactgcagcccccaccacaccgccctgaggcaggccatcctgtgctggggcgagctgatgaccctggccacctgggtgggcagcaacctggaggaccccgccagcagggagctggtggtgagctacgtgaacgtgaacatgggcctgaagatcaggcagctgctgtggttccacatcagctgcctgaccttcggcagggagaccgtgatcgagtacctggtgagcttcggcgtgtggatcaggaccccccccgcctacaggccccccaacgcccccatcctgagcaccctgcccgagaccaccgtggtgaggaggagggacaggggcaggagccccaggaggaggacccccagccccaggaggaggaggagccagagccccaggaggaggaggagccagagcagggagagccagtgcgggcccaaggccccatacacgagcaccctgctgcccccggagctgtccgagacccccaacgccacgcagccagaactcgccccggaagaccccgaggattcggccctcttggaggaccccgtggggacggtggcgccgcaaatcccaccaaactggcacatcccgtcgatccaggacgccgcgacgccttaccatcccccggccaccccgaacaacatgggcctgatcgccggcgcggtgggcggcagtctcctggcagccctggtcatttgcggaattgtgtactggatgcaccgccgcactcggaaagccccaaagcgcatacgcctcccccacatccgggaagacgaccagccgtcctcg caccagcccttgttttactaggDPolN amino MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRF acid sequenceRGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYY PolN underlinedAVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFR (SEQ ID NO: 16)MGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSA Signal peptideVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRA in italicsKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPPLSYQHFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGITRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHIPLHPAAMPGPKAPYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMHRRTRKAPKRIRLPHIREDDQPSSHQPLFY* gDPolN nucleicatggggggggctgccgccaggttgggggccgtgattttgttt acid sequencegtcgtcatagtgggcctccatggggtccgcggcaaatatgcc PolN underlinedttggcggatgcctctctcaagatggccgaccccaatcgcttt (SEQ ID NO: 17)cgcggcaaagaccttccggtcctggaccagctgaccgaccctccgggggtccggcgcgtgtaccacatccaggcgggcctaccggacccgttccagccccccagcctcccgatcacggtttactacgccgtgttggagcgcgcctgccgcagcgtgctcctaaacgcaccgtcggaggccccccagattgtccgcggggcctccgaagacgtccggaaacaaccctacaacctgaccatcgcttggtttcggatgggaggcaactgtgctatccccatcacggtcatggagtacaccgaatgctcctacaacaagtctctgggggcctgtcccatccgaacgcagccccgctggaactactatgacagcttcagcgccgtcagcgaggataacctggggttcctgatgcacgcccccgcgtttgagaccgccggcacgtacctgcggctcgtgaagataaacgactggacggagattacacagtttatcctggagcaccgagccaagggctcctgtaagtacgccctcccgctgcgcatccccccgtcagcctgcctctccccccaggcctaccagcagggggtgacggtggacagcatcgggatgctgccccgcttcatccccgagaaccagcgcaccgtcgccgtatacagcttgaagatcgccgggtggcacgggccccccctgagctaccagcacttcaggaagctgctgctgctggacgaggaggccggccccctggaggaggagctgcccaggctggccgacgagggcctgaacaggagggtggccgaggacctgaacctgggcaacctgaacgtgagcatcccctggacccacaaggtgggcaacttcaccggcctgtacagcagcaccgtgcccgtgttcaaccccgagtggcagacccccagcttccccaagatccacctgcaggaggacatcgtggacaggtgcaagcagttcgtgggtcccctgaccgtgaacgagaagaggaggctgaagctgatcatgcccgccaggttctaccccaacgtgaccaagtacctgcccctggacaagggcatcaagccctactaccccgagcacgccgtgaaccactacttccagaccaggcactacctgcacaccctgtggaaggccggcatcctgtacaagagggagaccaccaggagcgccagcttctgcggcagcccctacagctgggagcaggagctgcagcacggcagctgctggtggctgcagttcaggaacagcaagccctgcagcgagtactgcctgacccacctggtgaacctgctggaggactggggtccctgcgacgagcacggcgagcaccacatcaggatccccaggacccccgccagggtgaccggcggcgtgttcctggtggacaagaacccccacaacaccgccgagagcaggctggtggtggacttcagccagttcagcaggggcatcaccagggtgagctggcccaagttcgccgtgcccaacctgcagagcctgaccaacctgctgagcagcaacctgagctggctgagcctggacgtgagcgccgccttctaccacatccccctgcaccccgccgccatgcccgggcccaaggccccatacacgagcaccctgctgcccccggagctgtccgagacccccaacgccacgcagccagaactcgccccggaagaccccgaggattcggccctcttggaggaccccgtggggacggtggcgccgcaaatcccaccaaactggcacatcccgtcgatccaggacgccgcgacgccttaccatcccccggccaccccgaacaacatgggcctgatcgccggcgcggtgggcggcagtctcctggcagccctggtcatttgcggaattgtgtactggatgcaccgccgcactcggaaagccccaaagcgcatacgcctcccccacatccgggaagacgaccagccgtcctcgcaccagcccttgttt tactag gDPolC aminoMGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRF acid sequenceRGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYY PolC underlinedAVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFR (SEQ ID NO: 18)MGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSA Signal peptideVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRA in italicsKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPHLLVGSSGLSRYVARLSSNSRIINHQHGTMQNLHDSCSRNLYVSLLLLYKTFGRKLHLYSHPIILKTKRWGYSLNFMGYVIGSWGSLPQDHIIQKIKECFRKLPVNRPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQSKQAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDPSRGRLGLSRPLLRLPFRPTTGRTSLYAVSPSVGPKAPYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMHRR TRKAPKRIRLPHIREDDQPSSHQPLFYgDPolC nucleic atggggggggctgccgccaggttgggggccgtgattttgttt acid sequencegtcgtcatagtgggcctccatggggtccgcggcaaatatgcc PolC underlinedttggcggatgcctctctcaagatggccgaccccaatcgcttt (SEQ ID NO: 19)cgcggcaaagaccttccggtcctggaccagctgaccgaccctccgggggtccggcgcgtgtaccacatccaggcgggcctaccggacccgttccagccccccagcctcccgatcacggtttactacgccgtgttggagcgcgcctgccgcagcgtgctcctaaacgcaccgtcggaggccccccagattgtccgcggggcctccgaagacgtccggaaacaaccctacaacctgaccatcgcttggtttcggatgggaggcaactgtgctatccccatcacggtcatggagtacaccgaatgctcctacaacaagtctctgggggcctgtcccatccgaacgcagccccgctggaactactatgacagcttcagcgccgtcagcgaggataacctggggttcctgatgcacgcccccgcgtttgagaccgccggcacgtacctgcggctcgtgaagataaacgactggacggagattacacagtttatcctggagcaccgagccaagggctcctgtaagtacgccctcccgctgcgcatccccccgtcagcctgcctctccccccaggcctaccagcagggggtgacggtggacagcatcgggatgctgccccgcttcatccccgagaaccagcgcaccgtcgccgtatacagcttgaagatcgccgggtggcacgggccccacctgctggtgggcagcagcggcctgagcaggtacgtggccaggctgagcagcaacagcaggatcatcaaccaccagcacggcaccatgcagaacctgcacgacagctgcagcaggaacctgtacgtgagcctgctgctgctgtacaagaccttcggcaggaagctgcacctgtacagccaccccatcatcctgaagaccaagaggtggggctacagcctgaacttcatgggctacgtgatcggcagctggggcagcctgccccaggaccacatcatccagaagatcaaggagtgcttcaggaagctgcccgtgaacaggcccatcgactggaaggtgtgccagaggatcgtgggcctgctgggcttcgccgcccccttcacccagtgcggctaccccgccctgatgcccctgtacgcctgcatccagagcaagcaggccttcaccttcagccccacctacaaggccttcctgagcaagcagtacctgaacctgtaccccgtggccaggcagaggcccggcctgtgccaggtgttcgccgacgccacccccaccggctggggcctggccatgggccaccagaggatgaggggcaccttcgtggcccccctgcccatccacaccgccgagctgctggccgcctgcttcgccaggagcaggagcggcgccaagatcctgggcaccgacaacagcgtggtgetgageaggaagtacaccagcttcccctggctgctgggctgcgccgccaactggatcctgaggggcaccagcttcgtgtacgtgcccagcgccctgaaccccgccgacgaccccagcaggggcaggctgggcctgagcaggcccctgctgaggctgcccttcaggcccaccaccggcaggaccagcctgtacgccgtgagccccagcgtggggcccaaggccccatacacgagcaccctgctgcccccggagctgtccgagacccccaacgccacgcagccagaactcgccccggaagaccccgaggattcggccctcttggaggaccccgtggggacggtggcgccgcaaatcccaccaaactggcacatcccgtcgatccaggacgccgcgacgccttaccatcccccggccaccccgaacaacatgggcctgatcgccggcgcggtgggcggcagtctcctggcagccctggtcatttgcggaattgtgtactggatgcaccgccgcactcggaaagccccaaagcgcatacgcctcccccacatccgggaagacgaccagccgtcctcgcaccagcccttgttttactag HBV PolN v2HFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLPE amino acidWQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARF sequenceYPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGIL (SEQ ID NO:YKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYC 173)LTHLVNLLEDWGPCDEHGEHHIRIPRTPARVT HBV2 amino acidYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETT sequenceRSASFCGSPYSNEQELQHGSCWNLQFRNSKPCSEYCLTHLVN (SEQ ID NO:LLEDWGPCDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAES 174)RLWDFSQFSRGITRVSWPKFAVPNLQSLTNLLSSNLSVVLSL (Pol N DVQAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPT (italics)-Pol CGWGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILG (underlined)-TDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPAD Core)DVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTWRRRDR GR HBV3 amino acidHFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLPE sequenceWQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARF (SEQ ID NO:YPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGIL 175)YKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYC (Pol NLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVT QAFTFSPTYK (italics)-Pol CAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQRMR (underlined)-GTFVAPLPIHTAELLAACFARSRSGAKILGTDNSVVLSRKYT Core)SFPWLLGCAANWILRGTSFVYVPSALNPADDVGSNLEDPASRELWSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTWRRRDRGR HBV2 nucleictatctgccgctggataaaggcattaaaccgtattatccggaa acid sequencecatgcggtgaaccattattttcagacccgccattatctgcat (SEQ ID NO:accctgtggaaagcgggcattctgtataaacgcgaaaccacc 176)cgcagcgcgagcttttgcggcagcccgtatagctgggaacaggaactgcagcatggcagctgctggtggctgcagtttcgcaacagcaaaccgtgcagcgaatattgcctgacccatctggtgaacctgctggaagattggggaccgtgcgatgaacatggcgaacatcatattcgcattccgcgcaccccggcgcgcgtgaccggcggcgtgtttctggtggataaaaacccgcataacaccgcggaaagccgcctggtggtggattttagccagtttagccgcggcattacccgcgtgagctggccgaaatttgcggtgccgaacctgcagagcctgaccaacctgctgagcagcaacctgagctggctgagcctggatgtgcaggcgtttacctUagcccgacctataaagcgtttctgagcaaacagtatctgaacctgtatccggtggcgcgccagcgcccgggcctgtgccaggtgtttgcggatgcgaccccgaccggctggggcctggcgatgggccatcagcgcatgcgcggcacctttgtggcgccgctgccgattcataccgcggaactgctggcggcgtgctttgcgcgcagccgcagcggcgcgaaaattctgggcaccgataacagcgtggtgctgagccgcaaatataccagctttccgtggctgctgggctgcgcggcgaactggattctgcgcggcaccagctttgtgtatgtgccgagcgcgctgaacccggcggatgatgtgggcagcaacctggaagatccggcgagccgcgaactggtggtgagctatgtgaacgtgaacatgggcctgaaaattcgccagctgctgtggtttcatattagctgcctgacctttggccgcgaaaccgtgattgaatatctggtgagctttggcgtgtggattcgcaccccgccggcgtatcgcccgccgaacgcgccgattctgagcaccctgccggaaaccaccgtggtgcgccgccgcgatcggg gccgc HBV3 nucleiccattttcgcaaactgctgctgctggatgaagaagcgggaccg acid sequencectggaagaagaactgccgcgcctggcggatgaaggcctgaac (SEQ ID NO:cgccgcgtggcggaagatctgaacctgggcaacctgccggaa 177)tggcagaccccgagctttccgaaaattcatctgcaggaagatattgtggatcgctgcaaacagtttgtgggaccgctgaccgtgaacgaaaaacgccgcctgaaactgattatgccggcgcgcttttatccgaacgtgaccaaatatctgccgctggataaaggcattaaaccgtattatccggaacatgcggtgaaccattattttcagacccgccattatctgcataccctgtggaaagcgggcattctgtataaacgcgaaaccacccgcagcgcgagcttttgcggcagcccgtatagctgggaacaggaactgcagcatggcagctgctggtggctgcagtttcgcaacagcaaaccgtgcagcgaatattgcctgacccatctggtgaacctgctggaagattggggaccgtgcgatgaacatggcgaacatcatattcgcattccgcgcaccccggcgcgcgtgacccaggcgtttacctttagcccgacctataaagcgtttctgagcaaacagtatctgaacctgtatccggtggcgcgccagcgcccgggcctgtgccaggtgtttgcggatgcgaccccgaccggctggggcctggcgatgggccatcagcgcatgcgcggcacctttgtggcgccgctgccgattcataccgcggaactgctggcggcgtgctttgcgcgcagccgcagcggcgcgaaaattctgggcaccgataacagcgtggtgctgagccgcaaatataccagctttccgtggctgctgggctgcgcggcgaactggattctgcgcggcaccagctttgtgtatgtgccgagcgcgctgaacccggcggatgatgtgggcagcaacctggaagatccggcgagccgcgaactggtggtgagctatgtgaacgtgaacatgggcctgaaaattcgccagctgctgtggtttcatattagctgcctgacctttggccgcgaaaccgtgattgaatatctggtgagctttggcgtgtggattcgcaccccgccggcgtatcgcccgccgaacgcgccgattctgagcaccctgccggaaaccaccgtggtgcgccgccga gatcgaggccgc HBV2 PolN aminoYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETT acid sequenceRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVN (SEQ ID NO:LLEDWGPCDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAES 178)RLVVDFSQFSRGITRVSWPKFAVPNLQSLTNLLSSNLSWLSL DV HBV2 PolC aminoQAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGW acid sequenceGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTD (SEQ ID NO:NSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADD 179) HBV2 Core aminoVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRE acid sequenceTVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRG (SEQ ID NO: 180) RHBV3 PolN amino HFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLPE acid sequenceWQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARF (SEQ ID NO:YPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGIL 181)YKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVT HBV3 PolC aminoQAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGW acid sequenceGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTD (SEQ ID NO:NSWLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADD 182) HBV3 Core aminoVGSNLEDPASRELWSYVNVNMGLKIRQLLWFHISCLTFGRE acid sequenceTVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTWRRRDRG (SEQ ID NO: R 183)gD-HBV2 nucleic atggggggggctgccgccaggttgggggccgtgattttgttt acid sequencegtcgtcatagtgggcctccatggggtccgcggcaaatatgcc (SEQ ID NO:ttggcggatgcctctctcaagatggccgaccccaatcgcttt 184)cgcggcaaagaccttccggtcctggaccagctgaccgaccctccgggggtccggcgcgtgtaccacatccaggcgggcctaccggacccgttccagccccccagcctcccgatcacggtttactacgccgtgttggagcgcgcctgccgcagcgtgctcctaaacgcaccgtcggaggccccccagattgtccgcggggcctccgaagacgtccggaaacaaccctacaacctgaccatcgcttggtttcggatgggaggcaactgtgctatccccatcacggtcatggagtacaccgaatgctcctacaacaagtctctgggggcctgtcccatccgaacgcagccccgctggaactactatgacagcttcagcgccgtcagcgaggataacctggggttcctgatgcacgcccccgcgtttgagaccgccggcacgtacctgcggctcgtgaagataaacgactggacggagattacacagtttatcctggagcaccgagccaagggctcctgtaagtacgccctcccgctgcgcatccccccgtcagcctgcctctccccccaggcctaccagcagggggtgacggtggacagcatcgggatgctgccccgcttcatccccgagaaccagcgcaccgtcgccgtatacagcttgaagatcgccgggtggcacgggccctatctgccgctggataaaggcattaaaccgtattatccggaacatgcggtgaaccattattttcagacccgccattatctgcataccctgtggaaagcgggcattctgtataaacgcgaaaccacccgcagcgcgagcttttgcggcagcccgtatagctgggaacaggaactgcagcatggcagctgctggtggctgcagtttcgcaacagcaaaccgtgcagcgaatattgcctgacccatctggtgaacctgctggaagattggggaccgtgcgatgaacatggcgaacatcatattcgcattccgcgcaccccggcgcgcgtgaccggcggcgtgtttctggtggataaaaacccgcataacaccgcggaaagccgcctggtggtggattttagccagtttagccgcggcattacccgcgtgagctggccgaaatttgcggtgccgaacctgcagagcctgaccaacctgctgagcagcaacctgagctggctgagcctggatgtgcaggcgtttacctttagcccgacctataaagcgtttctgagcaaacagtatctgaacctgtatccggtggcgcgccagcgcccgggcctgtgccaggtgtttgcggatgcgaccccgaccggctggggcctggcgatgggccatcagcgcatgcgcggcacctttgtggcgccgctgccgattcataccgcggaactgctggcggcgtgctttgcgcgcagccgcagcggcgcgaaaattctgggcaccgataacagcgtggtgctgagccgcaaatataccagctttccgtggctgctgggctgcgcggcgaactggattctgcgcggcaccagctttgtgtatgtgccgagcgcgctgaacccggcggatgatgtgggcagcaacctggaagatccggcgagccgcgaactggtggtgagctatgtgaacgtgaacatgggcctgaaaattcgccagctgctgtggtttcatattagctgcctgacctttggccgcgaaaccgtgattgaatatctggtgagctttggcgtgtggattcgcaccccgccggcgtatcgcccgccgaacgcgccgattctgagcaccctgccggaaaccaccgtggtgcgccgccgcgatcggggccgcgggcccaaggccccatacacgagcaccctgctgcccccggagctgtccgagacccccaacgccacgcagccagaactcgccccggaagaccccgaggattcggccctcttggaggaccccgtggggacggtggcgccgcaaatcccaccaaactggcacatcccgtcgatccaggacgccgcgacgccttaccatcccccggccaccccgaacaacatgggcctgatcgccggcgcggtgggcggcagtctcctggcagccctggtcatttgcggaattgtgtactggatgcaccgccgcactcggaaagccccaaagcgcatacgcctcccccacatccgggaagacgaccagccgtcctcg caccagcccttgttttactaggD-HBV2 amino MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRF acid sequenceRGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYY (SEQ ID NO:AVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFR 185)MGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGITRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVQAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRGPKAPYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMHRRTRKAPKR IRLPHIREDDQPSSHQPLFY*gD-HBV3 nucleic atggggggggctgccgccaggttgggggccgtgattttgttt acid sequencegtcgtcatagtgggcctccatggggtccgcggcaaatatgcc (SEQ ID NO:ttggcggatgcctctctcaagatggccgaccccaatcgcttt 186)cgcggcaaagaccttccggtcctggaccagctgaccgaccctccgggggtccggcgcgtgtaccacatccaggcgggcctaccggacccgttccagccccccagcctcccgatcacggtttactacgccgtgttggagcgcgcctgccgcagcgtgctcctaaacgcaccgtcggaggccccccagattgtccgcggggcctccgaagacgtccggaaacaaccctacaacctgaccatcgcttggtttcggatgggaggcaactgtgctatccccatcacggtcatggagtacaccgaatgctcctacaacaagtctctgggggcctgtcccatccgaacgcagccccgctggaactactatgacagcttcagcgccgtcagcgaggataacctggggttcctgatgcacgcccccgcgtttgagaccgccggcacgtacctgcggctcgtgaagataaacgactggacggagattacacagtttatcctggagcaccgagccaagggctcctgtaagtacgccctcccgctgcgcatccccccgtcagcctgcctctccccccaggcctaccagcagggggtgacggtggacagcatcgggatgctgccccgcttcatccccgagaaccagcgcaccgtcgccgtatacagcttgaagatcgccgggtggcacgggccccattttcgcaaactgctgctgctggatgaagaagcgggaccgctggaagaagaactgccgcgcctggcggatgaaggcctgaaccgccgcgtggcggaagatctgaacctgggcaacctgccggaatggcagaccccgagctttccgaaaattcatctgcaggaagatattgtggatcgctgcaaacagtttgtgggaccgctgaccgtgaacgaaaaacgccgcctgaaactgattatgccggcgcgcttttatccgaacgtgaccaaatatctgccgctggataaaggcattaaaccgtattatccggaacatgcggtgaaccattattttcagacccgccattatctgcataccctgtggaaagcgggcattctgtataaacgcgaaaccacccgcagcgcgagcttttgcggcagcccgtatagctgggaacaggaactgcagcatggcagctgctggtggctgcagtttcgcaacagcaaaccgtgcagcgaatattgcctgacccatctggtgaacctgctggaagattggggaccgtgcgatgaacatggcgaacatcatattcgcattccgcgcaccccggcgcgcgtgacccaggcgtttacctttagcccgacctataaagcgtttctgagcaaacagtatctgaacctgtatccggtggcgcgccagcgcccgggcctgtgccaggtgtttgcggatgcgaccccgaccggctggggcctggcgatgggccatcagcgcatgcgcggcacctttgtggcgccgctgccgattcataccgcggaactgctggcggcgtgctttgcgcgcagccgcagcggcgcgaaaattctgggcaccgataacagcgtggtgctgagccgcaaatataccagctttccgtggctgctgggctgcgcggcgaactggattctgcgcggcaccagctttgtgtatgtgccgagcgcgctgaacccggcggatgatgtgggcagcaacctggaagatccggcgagccgcgaactggtggtgagctatgtgaacgtgaacatgggcctgaaaattcgccagctgctgtggtttcatattagctgcctgacctttggccgcgaaaccgtgattgaatatctggtgagctttggcgtgtggattcgcaccccgccggcgtatcgcccgccgaacgcgccgattctgagcaccctgccggaaaccaccgtggtgcgccgccgagatcgaggccgcgggcccaaggccccatacacgagcaccctgctgcccccggagctgtccgagacccccaacgccacgcagccagaactcgccccggaagaccccgaggattcggccctcttggaggaccccgtggggacggtggcgccgcaaatcccaccaaactggcacatcccgtcgatccaggacgccgcgacgccttaccatcccccggccaccccgaacaacatgggcctgatcgccggcgcggtgggcggcagtctcctggcagccctggtcatttgcggaattgtgtactggatgcaccgccgcactcggaaagccccaaagcgcatacgcctcccccacatccgggaagacgaccagccg tcctcgcaccagcccttgttttactaggD-HBV3 amino MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRF acid sequenceRGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYY (SEQ ID NO:AVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFR 187)MGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRAKGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPHFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLPEWQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVTQAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRGPKAPYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMHRRTRKAPKRIRLPHIREDDQP SSHQPLFY*

Embodiments

The following list of embodiments is intended to complement, rather thandisplace or supersede, the previous descriptions.

-   -   Embodiment 1. A hepatitis B virus (HBV) Core protein comprising        the amino acid sequence of SEQ ID NO: 6 or an immunogenic        fragment thereof.    -   Embodiment 2. The HBV Core protein of embodiment 1, wherein the        immunogenic fragment comprises any one of SEQ ID NOs: 20-54.    -   Embodiment 3. A hepatitis B virus (HBV) Core protein comprising        the amino acid sequence of SEQ ID NO: 180 or an immunogenic        fragment thereof, or the amino acid sequence of SEQ ID NO: 183        or an immunogenic fragment thereof.    -   Embodiment 4. A nucleic acid molecule encoding the HBV Core        protein of any one of embodiments 1-3.    -   Embodiment 5. The nucleic acid molecule of embodiment 4, wherein        the nucleic acid molecule comprises the nucleotide sequence of        SEQ ID NO: 7.    -   Embodiment 6. A vector comprising the nucleic acid molecule of        embodiment 4 or 5.    -   Embodiment 7. The vector of embodiment 6, wherein the vector is        an adenoviral vector.    -   Embodiment 8. The vector of embodiment 7, wherein the adenoviral        vector is an AdC6 vector or AdC7 vector.    -   Embodiment 9. A vaccine comprising the vector of any one of        embodiments 6-8.    -   Embodiment 10. A HBV polymerase N-terminal domain comprising the        amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment        thereof.    -   Embodiment 11. The HBV polymerase N-terminal domain of        embodiment 10, wherein the immunogenic fragment comprises any        one of SEQ ID NOs: 55-113.    -   Embodiment 12. A HBV polymerase N-terminal domain comprising the        amino acid sequence of SEQ ID NO: 178 or an immunogenic fragment        thereof, or the amino acid sequence of SEQ ID NO: 181 or an        immunogenic fragment thereof.    -   Embodiment 13. A HBV polymerase C-terminal domain comprising the        amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment        thereof.    -   Embodiment 14. The HBV polymerase C-terminal domain of        embodiment 13, wherein the immunogenic fragment comprises any        one of SEQ ID NOs: 114-172.    -   Embodiment 15. A HBV polymerase C-terminal domain comprising the        amino acid sequence of SEQ ID NO: 179 or an immunogenic fragment        thereof, or the amino acid sequence of SEQ ID NO: 182 or an        immunogenic fragment thereof.    -   Embodiment 16. A nucleic acid molecule encoding the HBV        polymerase of any one of embodiments 10-15.    -   Embodiment 17. The nucleic acid molecule of embodiment 16,        wherein the nucleic acid molecule comprises the nucleotide        sequence of SEQ ID NO: 9.    -   Embodiment 18. The nucleic acid molecule of embodiment 16,        wherein the nucleic acid molecule comprises the nucleotide        sequence of SEQ ID NO: 11.    -   Embodiment 19. A vector comprising the nucleic acid molecule of        any one of embodiments 16-18.    -   Embodiment 20. The vector of embodiment 19, wherein the vector        is an adenoviral vector.    -   Embodiment 21. The vector of embodiment 20, wherein the        adenoviral vector is an AdC6 vector or AdC7 vector.    -   Embodiment 22. A vaccine comprising the vector of any one of        embodiments 19-21.    -   Embodiment 23. A fusion protein comprising:        -   one or more of an HBV Core protein comprising the amino acid            sequence of SEQ ID NO: 6 or an immunogenic fragment thereof,            an HBV polymerase N-terminal domain comprising the amino            acid sequence of SEQ ID NO: 8 or an immunogenic fragment            thereof, and an HBV polymerase C-terminal domain comprising            the amino acid sequence of SEQ ID NO: 10 or an immunogenic            fragment thereof.    -   Embodiment 24. The fusion protein of embodiment 23, comprising:        -   (1) an HBV Core protein comprising the amino acid sequence            of SEQ ID NO: 6 or an immunogenic fragment thereof and an            HBV polymerase N-terminal domain comprising the amino acid            sequence of SEQ ID NO: 8 or an immunogenic fragment thereof;        -   (2) one or more of SEQ ID NOs: 20-54 (immunogenic fragments            of SEQ ID NO: 6) and one or more of SEQ ID NOs: 55-113            (immunogenic fragments of SEQ ID NO: 8);        -   (3) an HBV Core protein comprising the amino acid sequence            of SEQ ID NO: 6 or an immunogenic fragment thereof and an            HBV polymerase C-terminal domain comprising the amino acid            sequence of SEQ ID NO: 10 or an immunogenic fragment            thereof;        -   (4) one or more of SEQ ID NOs: 20-54 (immunogenic fragments            of SEQ ID NO: 6) and one or more of SEQ ID NOs: 114-172            (immunogenic fragments of SEQ ID NO: 10);        -   (5) an HBV polymerase N-terminal domain comprising the amino            acid sequence of SEQ ID NO: 8 or an immunogenic fragment            thereof and an HBV polymerase C-terminal domain comprising            the amino acid sequence of SEQ ID NO: 10 or an immunogenic            fragment thereof;        -   (6) one or more of SEQ ID NOs: 55-113 (immunogenic fragments            of SEQ ID NO: 8) and one or more of SEQ ID NOs: 114-172            (immunogenic fragments of SEQ ID NO: 10);        -   (7) an HBV Core protein comprising the amino acid sequence            of SEQ ID NO: 6 or an immunogenic fragment thereof, an HBV            polymerase N-terminal domain comprising the amino acid            sequence of SEQ ID NO: 8 or an immunogenic fragment thereof,            and an HBV polymerase C-terminal domain comprising the amino            acid sequence of SEQ ID NO: 10 or an immunogenic fragment            thereof; or        -   (8) one or more of SEQ ID NOs: 20-54 (immunogenic fragments            of SEQ ID NO: 6), one or more of SEQ ID NOs: 55-113            (immunogenic fragments of SEQ ID NO: 8), and one or more of            SEQ ID NOs: 114-172 (immunogenic fragments of SEQ ID NO:            10).    -   Embodiment 25. A fusion protein comprising:        -   an HBV polymerase N-terminal domain comprising the amino            acid sequence of SEQ ID NO: 178 or an immunogenic fragment            thereof, an HBV polymerase C-terminal domain comprising the            amino acid sequence of SEQ ID NO: 179 or an immunogenic            fragment thereof, and an HBV Core protein comprising the            amino acid sequence of SEQ ID NO: 180 or an immunogenic            fragment thereof.    -   Embodiment 26. The fusion protein of embodiment 25, comprising        the amino acid sequence of SEQ ID NO: 174.    -   Embodiment 27. A fusion protein comprising:        -   an HBV polymerase N-terminal domain comprising the amino            acid sequence of SEQ ID NO: 181 or an immunogenic fragment            thereof, an HBV polymerase C-terminal domain comprising the            amino acid sequence of SEQ ID NO: 182 or an immunogenic            fragment thereof, and an HBV Core protein comprising the            amino acid sequence of SEQ ID NO: 183 or an immunogenic            fragment thereof.    -   Embodiment 28. The fusion protein of embodiment 27, comprising        the amino acid sequence of SEQ ID NO: 175.    -   Embodiment 29. A fusion protein comprising:        -   an N-terminal herpes simplex virus (HSV) glycoprotein (gD)            sequence or a variant thereof;        -   an HBV Core protein comprising the amino acid sequence of            SEQ ID NO: 6 or an immunogenic fragment thereof, and        -   a C-terminal HSV gD sequence or a variant thereof.    -   Embodiment 30. The fusion protein of embodiment 29, wherein the        immunogenic fragment comprises any one of SEQ ID NOs: 20-54.    -   Embodiment 31. A fusion protein comprising:        -   an N-terminal HSV gD sequence or a variant thereof;        -   an HBV polymerase N-terminal domain comprising the amino            acid sequence of SEQ ID NO: 8 or an immunogenic fragment            thereof, and        -   a C-terminal HSV gD protein sequence or a variant thereof.    -   Embodiment 32. The fusion protein of embodiment 31, wherein the        immunogenic fragment comprises any one of SEQ ID NOs: 55-113.    -   Embodiment 33. A fusion protein comprising:        -   an N-terminal HSV gD sequence or a variant thereof;        -   an HBV polymerase C-terminal domain comprising the amino            acid sequence of SEQ ID NO: 10 or an immunogenic fragment            thereof, and        -   a C-terminal HSV gD protein sequence or a variant thereof.    -   Embodiment 34. The fusion protein of embodiment 33, wherein the        immunogenic fragment comprises any one of SEQ ID NOs: 114-172.    -   Embodiment 35. A fusion protein comprising:        -   an N-terminal HSV gD sequence or a variant thereof;        -   an HBV sequence comprising:        -   (1) an HBV Core protein comprising the amino acid sequence            of SEQ ID NO: 6 or an immunogenic fragment thereof and an            HBV polymerase N-terminal domain comprising the amino acid            sequence of SEQ ID NO: 8 or an immunogenic fragment thereof;        -   (2) one or more of SEQ ID NOs: 20-54 (immunogenic fragments            of SEQ ID NO: 6) and one or more of SEQ ID NOs: 55-113            (immunogenic fragments of SEQ ID NO: 8);        -   (3) an HBV Core protein comprising the amino acid sequence            of SEQ ID NO: 6 or an immunogenic fragment thereof and an            HBV polymerase C-terminal domain comprising the amino acid            sequence of SEQ ID NO: 10 or an immunogenic fragment            thereof;        -   (4) one or more of SEQ ID NOs: 20-54 (immunogenic fragments            of SEQ ID NO: 6) and one or more of SEQ ID NOs: 114-172            (immunogenic fragments of SEQ ID NO: 10);        -   (5) an HBV polymerase N-terminal domain comprising the amino            acid sequence of SEQ ID NO: 8 or an immunogenic fragment            thereof and an HBV polymerase C-terminal domain comprising            the amino acid sequence of SEQ ID NO: 10 or an immunogenic            fragment thereof;        -   (6) one or more of SEQ ID NOs: 55-113 (immunogenic fragments            of SEQ ID NO: 8) and one or more of SEQ ID NOs: 114-172            (immunogenic fragments of SEQ ID NO: 10);        -   (7) an HBV Core protein comprising the amino acid sequence            of SEQ ID NO: 6 or an immunogenic fragment thereof, an HBV            polymerase N-terminal domain comprising the amino acid            sequence of SEQ ID NO: 8 or an immunogenic fragment thereof,            and an HBV polymerase C-terminal domain comprising the amino            acid sequence of SEQ ID NO: 10 or an immunogenic fragment            thereof; or        -   (8) one or more of SEQ ID NOs: 20-54 (immunogenic fragments            of SEQ ID NO: 6), one or more of SEQ ID NOs: 55-113            (immunogenic fragments of SEQ ID NO: 8), and one or more of            SEQ ID NOs: 114-172 (immunogenic fragments of SEQ ID NO: 10)            and        -   a C-terminal HSV gD protein sequence or a variant thereof.    -   Embodiment 36. A fusion protein comprising:        -   an N-terminal HSV gD sequence or a variant thereof;        -   an HBV Core protein comprising the amino acid sequence of            SEQ ID NO: 180 or an immunogenic fragment thereof, or the            amino acid sequence of SEQ ID NO: 183 or an immunogenic            fragment thereof, and        -   a C-terminal HSV gD sequence or a variant thereof.    -   Embodiment 37. A fusion protein comprising:        -   an N-terminal HSV gD sequence or a variant thereof;        -   an HBV polymerase N-terminal domain comprising the amino            acid sequence of SEQ ID NO: 178 or an immunogenic fragment            thereof, or the amino acid sequence of SEQ ID NO: 181 or an            immunogenic fragment thereof, and        -   a C-terminal HSV gD protein sequence or a variant thereof.    -   Embodiment 38. A fusion protein comprising:        -   an N-terminal HSV gD sequence or a variant thereof;        -   an HBV polymerase C-terminal domain comprising the amino            acid sequence of SEQ ID NO: 179 or an immunogenic fragment            thereof, or the amino acid sequence of SEQ ID NO: 182 or an            immunogenic fragment thereof, and        -   a C-terminal HSV gD protein sequence or a variant thereof.    -   Embodiment 39. A fusion protein comprising:        -   an N-terminal HSV gD sequence or a variant thereof;        -   an HBV sequence comprising:        -   (1) an HBV polymerase N-terminal domain comprising the amino            acid sequence of SEQ ID NO: 178 or an immunogenic fragment            thereof, an HBV polymerase C-terminal domain comprising the            amino acid sequence of SEQ ID NO: 179 or an immunogenic            fragment thereof, and an HBV Core protein comprising the            amino acid sequence of SEQ ID NO: 180 or an immunogenic            fragment thereof, or        -   (2) an HBV polymerase N-terminal domain comprising the amino            acid sequence of SEQ ID NO: 181 or an immunogenic fragment            thereof, an HBV polymerase C-terminal domain comprising the            amino acid sequence of SEQ ID NO: 182 or an immunogenic            fragment thereof, and an HBV Core protein comprising the            amino acid sequence of SEQ ID NO: 183 or an immunogenic            fragment thereof, and        -   a C-terminal HSV gD protein sequence or a variant thereof.    -   Embodiment 40. The fusion protein of embodiment 39, wherein the        HBV sequence comprises an HBV polymerase N-terminal domain        comprising the amino acid sequence of SEQ ID NO: 178 or an        immunogenic fragment thereof, an HBV polymerase C-terminal        domain comprising the amino acid sequence of SEQ ID NO: 179 or        an immunogenic fragment thereof, and an HBV Core protein        comprising the amino acid sequence of SEQ ID NO: 180 or an        immunogenic fragment thereof.    -   Embodiment 41. The fusion protein of embodiment 40, wherein the        HBV sequence comprises the amino acid sequence of SEQ ID NO:        174.    -   Embodiment 42. The fusion protein of embodiment 39, wherein the        HBV sequence comprises an HBV polymerase N-terminal domain        comprising the amino acid sequence of SEQ ID NO: 181 or an        immunogenic fragment thereof, an HBV polymerase C-terminal        domain comprising the amino acid sequence of SEQ ID NO: 182 or        an immunogenic fragment thereof, and an HBV Core protein        comprising the amino acid sequence of SEQ ID NO: 183 or an        immunogenic fragment thereof    -   Embodiment 43. The fusion protein of embodiment 42, wherein the        HBV sequence comprises the amino acid sequence of SEQ ID NO:        175.    -   Embodiment 44. The fusion protein of any one of embodiments        29-43, wherein the N-terminal HSV gD sequence comprises the        amino acid sequence of SEQ ID NO: 12.    -   Embodiment 45. The fusion protein of any one of embodiments        29-43, wherein the N-terminal HSV gD sequence comprises amino        acid residues 26-269 of SEQ ID NO: 12.    -   Embodiment 46. The fusion protein of any one of embodiments        29-45, wherein the C-terminal HSV gD sequence comprises the        transmembrane domain of the HSV gD.    -   Embodiment 47. The fusion protein of any one of embodiments        29-46, wherein the C-terminal HSV gD sequence comprises the        amino acid sequence of SEQ ID NO: 13.    -   Embodiment 48. The fusion protein of any one of embodiments        29-47, wherein the fusion protein comprises the amino acid        sequence of any one of SEQ ID NO: 14 or an immunogenic fragment        thereof, SEQ ID NO: 16 or an immunogenic fragment thereof, or        SEQ ID NO: 18 or an immunogenic fragment thereof.    -   Embodiment 49. The fusion protein of any one of embodiments        39-47, wherein the fusion protein comprises the amino acid        sequence of SEQ ID NO: 185.    -   Embodiment 50. The fusion protein of any one of embodiments        39-47, wherein the fusion protein comprises the amino acid        sequence of SEQ ID NO: 187.    -   Embodiment 51. A nucleic acid molecule encoding the fusion        protein of any one of embodiments 23-50.    -   Embodiment 52. The nucleic acid molecule of embodiment 51,        wherein the nucleic acid molecule comprises the nucleotide        sequence of any one of SEQ ID NOs: 15, 17, or 19.    -   Embodiment 53. The nucleic acid molecule of embodiment 51,        wherein the nucleic acid molecule comprises the nucleotide        sequence of SEQ ID NO: 176.    -   Embodiment 54. The nucleic acid molecule of embodiment 51,        wherein the nucleic acid molecule comprises the nucleotide        sequence of SEQ ID NO: 177.    -   Embodiment 55. The nucleic acid molecule of embodiment 51,        wherein the nucleic acid molecule comprises the nucleotide        sequence of SEQ ID NO: 184.    -   Embodiment 56. The nucleic acid molecule of embodiment 51,        wherein the nucleic acid molecule comprises the nucleotide        sequence of SEQ ID NO: 186.    -   Embodiment 57. A vector comprising the nucleic acid molecule of        any one of embodiments 51-56.    -   Embodiment 58. The vector of embodiment 57, wherein the vector        is an adenoviral vector.    -   Embodiment 59. The vector of embodiment 58, wherein the        adenoviral vector is an AdC6 vector or AdC7 vector.    -   Embodiment 60. A vaccine comprising the vector of any one of        embodiments 57-59.    -   Embodiment 61. A method of inducing an immune response to HBV in        a subject, the method comprising providing to the subject an        effective amount of the fusion protein of any one of embodiments        23-50, the nucleic acid molecule of any one of embodiments        51-56, the vector of any one of embodiments 57-59, or the        vaccine of embodiment 60 to thereby induce an immune response to        HBV.    -   Embodiment 62. The method of embodiment 61, wherein the vaccine        comprises an AdC6 vector comprising a fusion protein comprising        the amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18,        or an immunogenic fragment thereof.    -   Embodiment 63. The method of embodiment 62, further comprising        providing to the subject, subsequent to providing the vaccine        comprising the AdC6 vector, a vaccine comprising an AdC7 vector        comprising a fusion protein comprising the amino acid sequence        of any one of SEQ ID NOs: 14, 16, or 18, or an immunogenic        fragment thereof.    -   Embodiment 64. The method of embodiment 61, wherein the vaccine        comprises an AdC7 vector comprising a fusion protein comprising        the amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18,        or an immunogenic fragment thereof.    -   Embodiment 65. The method of embodiment 64, further comprising        providing to the subject, subsequent to providing the vaccine        comprising the AdC7 vector, a vaccine comprising an AdC6 vector        comprising a fusion protein comprising the amino acid sequence        of any one of SEQ ID NOs: 14, 16, or 18, or an immunogenic        fragment thereof.    -   Embodiment 66. The method of embodiment 61, wherein the vaccine        comprises an AdC6 vector comprising a fusion protein comprising        the amino acid sequence of SEQ ID NO: 185.    -   Embodiment 67. The method of embodiment 66, further comprising        providing to the subject, subsequent to providing the vaccine        comprising the AdC6 vector, a vaccine comprising an AdC7 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 185.    -   Embodiment 68. The method of embodiment 61, wherein the vaccine        comprises an AdC7 vector comprising a fusion protein comprising        the amino acid sequence of SEQ ID NO: 185.    -   Embodiment 69. The method of embodiment 68, further comprising        providing to the subject, subsequent to providing the vaccine        comprising the AdC7 vector, a vaccine comprising an AdC6 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 185.    -   Embodiment 70. The method of embodiment 61, wherein the vaccine        comprises an AdC6 vector comprising a fusion protein comprising        the amino acid sequence of SEQ ID NO: 187.    -   Embodiment 71. The method of embodiment 70, further comprising        providing to the subject, subsequent to providing the vaccine        comprising the AdC6 vector, a vaccine comprising an AdC7 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 187.    -   Embodiment 72. The method of embodiment 61, wherein the vaccine        comprises an AdC7 vector comprising a fusion protein comprising        the amino acid sequence of SEQ ID NO: 187.    -   Embodiment 73. The method of embodiment 72, further comprising        providing to the subject, subsequent to providing the vaccine        comprising the AdC7 vector, a vaccine comprising an AdC6 vector        comprising a fusion protein comprising the amino acid sequence        of SEQ ID NO: 187.    -   Embodiment 74. The method of any one of embodiments 61-73,        wherein the amino acid sequence of any one of SEQ ID NOs: 14,        16, 18, 185, or 187, or an immunogenic fragment thereof, does        not contain the N-terminal 25 amino acid signal peptide.

What is claimed:
 1. A nucleic acid molecule comprising a nucleotidesequence encoding a hepatitis B virus (HBV) polymerase N-terminal domaincomprising the amino acid sequence of SEQ ID NO: 178 or an immunogenicfragment thereof.
 2. The nucleic acid molecule of claim 1, comprisingthe nucleotide sequence of nucleic acids 1-510 of SEQ ID NO:
 176. 3. Avector comprising the nucleic acid molecule of claim
 2. 4. The vector ofclaim 3, wherein the vector is an adenoviral vector.
 5. The vector ofclaim 4, wherein the adenoviral vector is an AdC6 vector or AdC7 vector.6. A vaccine comprising the vector of claim
 5. 7. A HBV polymeraseN-terminal domain comprising the amino acid sequence of SEQ ID NO: 178or an immunogenic fragment thereof.
 8. A nucleic acid moleculecomprising a nucleotide sequence encoding a HBV polymerase C-terminaldomain comprising the amino acid sequence of SEQ ID NO: 179 or animmunogenic fragment thereof.
 9. The nucleic acid molecule of claim 8,comprising the nucleotide sequence of nucleic acids 511-885 of SEQ IDNO:
 176. 10. A vector comprising the nucleic acid molecule of claim 9.11. The vector of claim 10, wherein the vector is an adenoviral vector.12. The vector of claim 11, wherein the adenoviral vector is an AdC6vector or AdC7 vector.
 13. A vaccine comprising the vector of claim 12.14. A HBV polymerase C-terminal domain comprising the amino acidsequence of SEQ ID NO: 179 or an immunogenic fragment thereof.
 15. Anucleic acid molecule comprising a nucleotide sequence encoding a HBVCore protein comprising the amino acid sequence of SEQ ID NO: 180 or animmunogenic fragment thereof.
 16. The nucleic acid molecule of claim 15,comprising the nucleotide sequence of nucleic acids 886-1140 of SEQ IDNO:
 176. 17. A vector comprising the nucleic acid molecule of claim 16.18. The vector of claim 17, wherein the vector is an adenoviral vector.19. The vector of claim 18, wherein the adenoviral vector is an AdC6vector or AdC7 vector.
 20. A vaccine comprising the vector of claim 19.21. A HBV Core protein comprising the amino acid sequence of SEQ ID NO:180 or an immunogenic fragment thereof.